Opiates such as morphine are still the best analgesic choice in the treatment of chronic and serious pain. The clinical utility of opiates is limited by adaptive changes in the nervous system occurring after prolonged or repeated drug administration. These adaptations are believed to contribute to physiological tolerance and dependence to opiates. All of these adaptive changes are initiated by the binding of opiate drugs to opioid receptors that are also activated by endogenous opioid neuropeptides. The µ-opioid receptor (MOR1) is of primary importance for mediating analgesic and addictive effects of clinically important opiate drugs. Understanding the mechanisms of MOR1 regulation is a key step to develop drugs and/or therapy, which result in effective analgesia without the detrimental adaptive responses. There is increasing evidence that receptor-associated proteins modulate the signal transduction of MOR1. Several proteins which interact with MOR1 including the membrane glycoprotein M6a (M6a) were previously identified by our group using a yeast two-hybrid assay. To confirm the protein-protein interactions in mammalian cells, the proteins were tagged with bioluminescent/fluorescent epitopes and their interactions were assayed by a bioluminescence resonance energy transfer (BRET) technique. Of all proteins investigated, M6a showed the strongest interaction with MOR1. This interaction was also confirmed by co-immunoprecipitation experiments. Furthermore, the transmembrane domains of MOR1 and M6a that are important for the interaction were characterized. In addition, we demonstrated the interaction of M6a with a number of GPCRs, suggesting that M6a might play a general role in the regulation of GPCRs. M6a is a member of the proteolipid protein (PLP)/DM20 family of unclear function. Double in situ hybridization showed a widespread co-expression between MOR1 and M6a in many regions of rat brain. In addition, in transfected HEK293 cells, MOR1 co-internalizes with M6a, and then co-recycles to cell surface by recycling endosomes. This is associated with an augmented internalization and recycling of the µ-opioid receptor. In HEK293 cells, a physiological role of endogenous M6a in MOR1 internalization was revealed since over-expression of M6a dominant negative mutants prevents agonist-mediated endocytosis of MOR1. In addition, by enhancing receptor recycling M6a decreases receptor degradation in lysosomes, consistent with the observed decrease in down-regulation of MOR1. M6a also binds to and co-internalizes with the d-opioid receptor (DOR1). The interaction between DOR1 and M6a directs receptor post-endocytotic sorting into recycling pathway, which further provides support for a role of M6a in receptor recycling. M6a-augmented MOR1 trafficking results in decreased receptor desensitization. Importantly, while the functional property of M6a on the trafficking and signal regulation of the µ-opioid receptor has been established in HEK293 cells, our studies in primary cultured neurons suggest a similar function also in native neurons. Taken together, M6a might function as an adaptor protein in receptor trafficking, and be involved in reducing the development of opiate tolerance. Therefore, our work revealed a new function of the PLP/DM20 glycoprotein family other than its structural role in myelination.