Regulation of intracellular trafficking of opioid receptors

The mu- and delta-opioid receptors (MOR, DOR) differ significantly in their intracellular trafficking. MORs recycle back to the cell surface upon agonist treatment, whereas most internalized DORs are targeted to lysosomes for degradation. The mechanism determining the specificity of opioid receptor trafficking after endocytosis is still unclear. By exchanging the carboxyl tail of MOR and DOR and expressing the receptor chimeras in mouse neuroblastoma Neuro2A (N2A) cells, it can be demonstrated that the carboxyl tail is not the sole determinant in directing the intracellular trafficking. Deletion of the dileucine motif (Leu245Leu246) within the 3rd intracellular loop of DOR, or the mutation of Leu 245 to Met slowed the lysosomal targeting of DOR. Meanwhile the mutation of Met264 to Leu increased the rate of agonistinduced receptor internalization and lysosomal targeting of the wild type and the DOR carboxyl tail chimera of the MOR. Truncation after amino acid Leu387 (HA-DOR/MTD387) significantly decreased opioid receptor recycling. Further deletion and mutagenesis analyses demonstrated that Leu387, Asn389, Leu390 and Leu397 all played critical roles in the opioid receptor recycling. Since Rab GTPases mediate agonist-induced trafficking of many G protein-coupled receptors (GPCRs), we investigated the identities and evaluated the roles of recycling motifs within MOR carboxyl tail in Rab11a-mediated receptor recycling. As we expect, HA-DOR/MT was sorted to the Rab11a positive vesicles after etorphine treatment. Overexpression of wild-type Rab11a significantly increased HA-DOR/MT recycling while dominant negative Rab11aS25N mutant inhibited receptor recycling after internalization and promoted down-regulation. Interestingly, HA-DOR/MTD387 still targeted to the Rab11a containing vesicles, but overexpression of wild type Rab11a could not accelerate HA-DOR/MTD387 recycling. Leu387, Asn389, and Leu390 all played critical roles in the Rab11a-dependent receptor recycling. Together, these studies suggest interplay between a di-leucine motif and the carboxyl tail in the lysosomal targeting of the receptor. Multiple motifs within MOR carboxyl tail determine correct distribution of internalized receptors to Rab11a sorting endosomes that eventually leads to recycling of the receptor. These studies provide new insights into the mechanisms underlying the opioid receptor downregulation and recycling and provide a basis to identify other proteins and motifs important in the opioid receptor intracellular trafficking.