Brain region-specific N-glycosylation and lipid rafts association of the rat mu opioid receptor
Section snippets
Materials and methods
Materials. [3H]Diprenorphine (58 Ci/mmole) and [35S]guanosine 5-(γ-thio)triphosphate (GTPγS) (1250 Ci/mmole) were purchased from Perkin-Elmer Co. (Boston, MA). Lectin from Triticum vulgaris (wheat germ agglutinin/WGA)-Agarose and methyl-β-cyclodextrin (MCD) were purchased from Sigma Co. (St Louis, MO). Anti-GM1 polyclonal antibody and PANSORBIN was purchased from Calbiochem (San Diego, CA). HA.11 was a product of Covance (Cumberland, VA). Anti-μC is a rabbit polyclonal anti-MOR antibody against
Western blotting of the MOR in CHO-HA-rMOR cells and in brains
For CHO-HA-rMOR cells, anti-μC-labeled proteins migrated as a major broad band with a median Mr of 78 kDa and a minor lower band of Mr 52 kDa (Fig. 1A, left panel), which are similar to the bands labeled by HA.11 (Fig. 1A, right panel). Both antibodies detected no specific bands in either CHO-FLAG-hKOR or CHO-FLAG-mDOR cells (Fig. 1A).
To detect endogenous MOR, CPu, thalamus, and cerebellum were dissected from mouse brains of wild-type and MOR-knockout (K/O) littermates, and membranes were
Discussion
To the best of our knowledge, this is the first report to show that the post-translational modification of a GPCR in the brain by N-glycosylation is region-specific. This is also the first demonstration that a membrane protein is associated with lipid rafts to varying degrees in different brain areas.
Acknowledgment
This work was supported by National Institutes of Health Grants: R01 DA17302 and P30 DA13429.
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Cited by (26)
The Life Cycle of the Mu-Opioid Receptor
2021, Trends in Biochemical SciencesCitation Excerpt :There are five asparagine residues located at the N-terminal of the MOR that can potentially be glycosylated (N-glycosylation). Interestingly, the MOR has been suggested to have brain-region specific glycosylation levels [55]. As mentioned above, the A118G polymorphism of the OPRM1 gene eliminates Asn40 and, therefore, a key site of glycosylation.
Post-translational Modifications of Opioid Receptors
2020, Trends in NeurosciencesCitation Excerpt :However, these results were not reproducible in COS cells [42]; this could be due to variation of the glycan profile between different cell lines [54]. The extent of N-glycosylation of MOR also differs among mouse brain regions; for example, the level of glycosylation is higher in the thalamus in comparison with the striatum [55]. Taken together, as in the case of other GPCRs, OR glycosylation has been described to modulates the dynamics of the steady-state levels of the receptor at the cell surface and consequently, of protein abundance [37,39,56,57].
Reduced expression of the mu opioid receptor in some, but not all, brain regions in mice with OPRM1 A112G
2012, NeuroscienceCitation Excerpt :Indeed, Matsuhashi et al. (2003) reported region-specific expression of glycotransferases in adult mouse brain. Thus, we hypothesize that the brain area-specific impact of A112G on MOPR expression may be due to differences in glycan content changes of the MOPR induced by this substitution across brain regions (Huang et al., 2008). This hypothesis remains to be investigated.
Endogenous opiates and behavior: 2008
2009, PeptidesNeuropeptide FF-sensitive confinement of mu opioid receptor does not involve lipid rafts in SH-SY5Y cells
2008, Biochemical and Biophysical Research CommunicationsCitation Excerpt :A possible explanation for this discrepancy could be that the physicochemical properties of the MOP receptor do not favor its association with lipid rafts fractions but that it is still loosely associated with this compartment by specific protein/lipid or protein/protein interactions that resist detergent-free extraction. The necessity for these specific interactions could also explain why lipid rafts association and cholesterol dependency of the MOP receptor is region-specific in the rat brain [16]. A stable SH-SY5Y cell line was also constructed in order to study the distribution of the human NPFF2 receptor fused to YFP at its C-terminus.