Membrane glycoprotein M6A promotes mu-opioid receptor endocytosis and facilitates receptor sorting into the recycling pathway.
The interaction of mu-opioid receptor (MOPr) with the neuronal membrane glycoprotein M6a is known to facilitate MOPr endocytosis in human embryonic kidney 293 (HEK293) cells. To further study the role of M6a in the post-endocytotic sorting of MOPr, we investigated the agonist-induced co-internalization of MOPr and M6a and protein targeting after internalization in HEK293 cells that co-expressed HA-tagged MOPr and Myc-tagged M6a. We found that M6a, MOPr, and Rab 11, a marker for recycling endosomes, co-localized in endocytotic vesicles, indicating that MOPr and M6a are primarily targeted to recycling endosomes after endocytosis. Furthermore, co-expression of M6a augmented the post-endocytotic sorting of delta-opioid receptors into the recycling pathway, indicating that M6a might have a more general role in opioid receptor post-endocytotic sorting. The enhanced post-endocytotic sorting of MOPr into the recycling pathway was accompanied by a decrease in agonist-induced receptor down-regulation of M6a in co-expressing cells. We tested the physiological relevance of these findings in primary cultures of cortical neurons and found that co-expression of M6a markedly increased the translocation of MOPrs from the plasma membrane to intracellular vesicles at steady state and significantly enhanced both constitutive and agonist-induced receptor endocytosis. In conclusion, our results strongly indicate that M6a modulates MOPr endocytosis and post-endocytotic sorting and has an important role in receptor regulation.
Department of Pharmacology and Toxicology, Otto-von-Guericke University, Leipziger Str. 44, 39120 Magdeburg, Germany.
Ying-Jian
YJ
Liang
Dai-Fei
DF
Wu
Ralf
R
Stumm
Volker
V
Höllt
Thomas
T
Koch
D002460:Cell Line; D015536:Down-Regulation; D004705:Endocytosis; D006801:Humans; D008562:Membrane Glycoproteins / Q000032:analysis / Q000378:metabolism; D008780:Methyltransferases / Q000032:analysis / Q000378:metabolism*; D009474:Neurons; D021381:Protein Transport; D017450:Receptors, Opioid, mu / Q000032:analysis / Q000378:metabolism*; D022161:Transport Vesicles / Q000737:chemistry; D020691:rab GTP-Binding Proteins / Q000032:analysis / Q000378:metabolism; D000099193:rab11 GTP-Binding Proteins
D008562:Membrane Glycoproteins; D008780:Methyltransferases; D017450:Receptors, Opioid, mu; D020691:rab GTP-Binding Proteins; D000099193:rab11 GTP-Binding Proteins; C485764:OPRM1 protein, human; C468275:METTL3 protein, human