Study On The μ-opioid Receptor Internalization

Opioid receptor of the subfamily of GPCRs,which serves as the principle physiological target for endogeneous opioid peptides and most clinically important opioid analgesics,modulates a number of physiological processes including pain, stress,and immune responses.To investigate the functional regulation of opioid receptor will bring cell biological and pharmacological mechanisms to opioid tolorence.Agonist-induced phosphorylation,internalization and intracellular trafficking of G-protein coupled receptors(GPCRs) are critical in regulating both cellular responsiveness and signal transduction.Many studies have suggested that phosphorylation of MOR modulates opioid receptor responsiveness,contributes to receptor desensitization,resensitization and opioid tolerance.However,the role of MOR phosphorylation in the regulation of its vesicular trafficking has not been clarity. The current study investigated the role of receptor phosphorylation in the regulation of agonist-induced internalization and intracellular trafficking of ratμ-opioid receptor (MOR) in HEK293 cell line.Our results showed that:1.After agonist stimulation,the internalization of a mutant MOR that lacks the C-terminal residues after Asn³⁶²(MOR362T) was greatly decreased,whereas a C-terminal phosphorylation sites-mutated MOR(MOR3A) which is deficient in agonist-induced phosphorylation internalized at a level comparable to that of the wild-type receptor.The wild-type MOR,MOR362T and MOR3A colocalized with Rab5-positive vesicles after their internalization.Furthermore,the constitutive-active and dominant-negative Rab5 mutants significantly increased or impeded the internalization of all these receptors,respectively.These data indicated that the carboxyl-terminal structure was important for the internalization of MOR,and phosphorylation of MOR was not essential for the Rab5-mediated internalization pathway.2.The recycling of the carboxyl-terminal truncated mutant(MOR362T) was greatly decreased,whereas the phosphorylation sites-mutated MOR(MOR3A) recycled back to the membrane at a level comparable to that of the wild-type receptor, however,interestingly at a slower recycling rate,suggesting that the carboxyl-terminal structure was critical for MOR recycling and missing this stucture missorted the receptor to the degradative pathway,whereas the phosphorylation of MOR only regulated the recycling kinetics of MOR.3.The recycling of the wild-type MOR was significantly impaired by overexpression of dominant-negative mutants and siRNA of both Rab4 and Rab 11, whereas the recycling of the phosphorylation-deficient mutant(MOR3A) was only inhibited by the dominant-negative mutant and siRNA of Rab11,suggesting that the recycling of nonphosphorylated MOR was exclusively via Rab11-mediated pathway.4.The efficacious inhibitor of phosphotases PP1/PP2A(okadaic acid ) significantly decreased the dephosphorylation and recycling of the wild-type MOR, whereas had no detectable effect on those of MOR3A.Additionally, immunofluorescent data showed that phosphorylated MOR was detained in Rab5-and Rab4-,but not Rab-11 positive vesicles.These observations suggested that the dephosphorylation of MORs mediated by PP1/PP2A was critical for the recycling of phosphorylated MOR,and further indiccated that the recycling of phosphorylated MOR was related with the Rab4 mediated vesicular trafficking pathway.In conclusion,our data establish that although carboxyl-terminal structure is important for the internalization and recycling of MOR,phosphorylation of MOR only regulates its recycling.Furthermore,both phosphorylated and nonphosphorylated MOR internalize via Rab5-dependent pathway after agonist stimulation,whereas the phosphorylated and nonphosphorylated MORs recycle through distinct vesicular trafficking pathways mediated by Rab4 and Rab11, respectively,which may ultimately lead to differential cellular responsiveness or downstream signaling.