Over the past several years, studies have highlighted the δ-opioid receptor (DOPr) as a promising therapeutic target for chronic pain management. While exhibiting milder undesired effects than most currently prescribed opioids, its specific agonists elicit effective analgesic responses in numerous animal models of chronic pain, including inflammatory, neuropathic, diabetic, and cancer-related pain. However, as compared with the extensively studied μ-opioid receptor, the molecular mechanisms governing its trafficking remain elusive. Recent advances have denoted several significant particularities in the regulation of DOPr intracellular routing, setting it apart from the other members of the opioid receptor family. Although they share high homology, each opioid receptor subtype displays specific amino acid patterns potentially involved in the regulation of its trafficking. These precise motifs or "barcodes" are selectively recognized by regulatory proteins and therefore dictate several aspects of the itinerary of a receptor, including its anterograde transport, internalization, recycling, and degradation. With a specific focus on the regulation of DOPr trafficking, this review will discuss previously reported, as well as potential novel trafficking barcodes within the opioid and nociceptin/orphanin FQ opioid peptide receptors, and their impact in determining distinct interactomes and physiological responses.

The complex and dynamic interplay between internalization, anterograde transport, recycling and degradation determines the density of functional G protein-coupled receptors (GPCRs) at the cell surface and, consequently, the magnitude of their associated physiological responses. As opposed to most members of the GPCR superfamily, the delta opioid receptor (DOP) is only weakly expressed at the neuronal plasma membrane, thus representing a critical limitation for its use as a therapeutic target. Although DOP appears as a promising candidate for the development of better-tolerated analgesics, the molecular and cellular mechanisms underlying the regulation of its cell surface expression remain poorly characterized. This work investigates the constitutive ( i.e. ligand-independent) trafficking of DOP, an understudied cellular process potentially involved in the control of plasma membrane-localized receptors. In HEK293 cells stably expressing Flag-tagged DOP, we first confirmed that this GPCR is constitutively internalized through a clathrin-dependent and b-arrestin-independent mechanism. Immunofluorescence experiments with selected Rab protein isoforms indicated that internalized DOP was mainly colocalized with the early endosome marker Rab5, as well as the rapid recycling endosome marker Rab4. Co-transfection with Rab5 dominant-negative mutant inhibited the intracellular distribution of the receptor, indicating that its constitutive endocytosis is Rab5-dependent. DOP cell surface expression and ligand-induced signaling were also significantly reduced following Rab4-specific DsiRNA treatments, suggesting a role for this small GTPase in the regulation of DOP constitutive recycling. Mapping of the major region of interaction between DOP and both Rabs revealed that Rab4 binds the third intracellular loop of DOP, whereas Rab5 seems to preferentially interact with the distal region of the C-terminal end of DOP. Altogether, these results show for the first time that DOP constitutive internalization and recycling are critical to maintain its cell surface bioavailability and responsiveness to agonists.

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