Membrane glycoprotein M6A promotes μ-opioid receptor endocytosis and facilitates receptor sorting into the recycling pathway

Abstract

The interaction of μ-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 δ-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.

Induction of axon growth arrest without growth cone collapse through the N‐terminal region of four‐transmembrane glycoprotein M6a

“…Thus, this domain might interact with some molecules responsible for the outgrowth arrest. Interestingly, a recent study reported that M6a binds to the seven-transmembrane l-opioid receptor via the transmembrane domains, and regulates distribution of these proteins onto the cell membrane surface (Wu et al, 2007;Liang et al, 2008), suggesting a general implication of M6a for recruitment and scaffolding function. Identifying M6a interactors on the growth cone edge may pave the way for understanding the unique axon behavior induced by the anti-M6a mAbs.…”

Molecular Pharmacology ofδ-Opioid Receptors

“…The next category of DOPr-interacting proteins corresponds to those involved in receptor desensitization and trafficking. Some of these proteins, such as GRKs and barrs, interact with the receptor after its stimulation (see sections VII.A and VII.B for details), whereas others like GASPs Simonin et al, 2004) and glycoprotein M6a (Wu et al, 2007;Liang et al, 2008) associate constitutively (Fig. 2).…”

“…Glycoprotein M6a is a membrane protein with four TMHs, two of which (the third and fourth) directly interact with TMH4, TMH5, and TMH6 of the receptor (Wu et al, 2007). M6a overexpression was shown to redirect DOPr to the recycling path (Liang et al, 2008), but it is not yet known whether it has this same effect at endogenous expression levels. NHERF also interacts with DOPr, although with much lower affinity than KOPr (Huang et al, 2004).…”

Actin-Independent Behavior and Membrane Deformation Exhibited by the Four-Transmembrane Protein M6a

“…Among them, we can acknowledge their potential involvement in membrane dynamics. More specifically, heterologous expression analyses have shown that M6a and M6b affect membrane trafficking and cell surface expression of G protein-coupled receptors such as the μ-opioid receptor and the serotonin transporter [21], [22], [23]. As the most abundant protein in the myelin membrane, PLP/DM20 is synthesized by oligodendrocytes, regulates the regularly-spaced apposition of myelin membranes, and maintains their stability [24], [25], [26].…”