Opioid receptor desensitization and endocytosis are critical steps in opioid tolerance. After opioid binding, the receptor is phosphorylated by G protein-coupled receptor kinases (GRKs) or second messenger kinases. The binding of ß-arrestins to the phosphorylated receptor leads to receptor uncoupling from G-proteins, which causes receptor desensitization. The ligand-receptor complex is internalized via clathrin coated vesicles. These vesicles soon shed their clathrin coat and become endosomes. The receptor is either recycled to the plasma membrane or degraded in the lysosome with the ligand. To identify components of this endocytotic machinery, we searched for the mu opioid receptor (MOR1) interacting proteins that could modulate trafficking and signaling of the receptor. We used different portions of the mu opioid receptors as baits to screen a rat brain cDNA library by a yeast two hybrid assay. A series of proteins were identified which might be involved in the receptor trafficking: like heat shock cognate protein 70, synaptophysin, membrane glycoprotein M6a and phospholipase D2. In the yeast two hybrid screen Hsc70 interacts with the carboxyl terminus of MOR1; Synaptophysin binds to the 3rd intracellular loop of MOR1; M6a interacts with the carboxyl terminus of splice variant MOR1B; PLD2 (PX domain) interacts with the carboxyl termini of MOR1 and MOR1B. The interaction between MOR1 and the three of the above four proteins (PLD2, M6a, synaptophysin) was confirmed in HEK 293 cells by Bioluminescence Resonance Energy Transfer (BRET) assay and/or coimmunoprecipitation experiment. The interaction was proved to be agonist-independent. Furthermore, the putative functions of these proteins on MOR1 trafficking were investigated. As the major integral membrane glycoprotein in the presynaptic vesicles of neurons, synaptophysin is known to interact with dynamin. Coexpression of MOR1 and synaptophysin resulted in constitutive internalization of the receptor. These results suggest that the association of MOR1 with synaptophysin plays a role in MOR1 trafficking by targeting dynamin to the presynaptic membrane. M6a is a membrane glycoprotein with high abundance in the central nervous system (CNS) of unclear function. After coexpression in HEK 293 cells, MOR1 showed strict colocalization with M6a both in the membrane and in endosome-like structures. The basal endocytotic rate of MOR1 was enhanced by M6a. M6a was cointernalized with the receptor after opioid agonist DAMGO treatment. DAMGO which promotes receptor internalization activates PLD2 in HEK 293 cells coexpressing MOR1 and PLD2. In contrast, morphine, an opioid alkaloid which fails to induce receptor internalization, does not activate PLD2. This leads to the hypothesis that the ability of agonist to induce endocytosis is linked to their ability to activate PLD2. It was further demonstrated that after agonist binding, PLD2 is stimulated by MOR1 in an ARF-dependent manner. The resultant acidic phospholipid-enriched membrane facilitates the budding and scission of clathrin coated vesicles. In conclusion, the identified proteins should help to elucidate the molecular mechanisms of opioid receptor tolerance.