Brain region-specific N-glycosylation and lipid rafts association of the rat mu opioid receptor

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Abstract

The mu opioid receptor (MOR) in the rat and mouse caudate putamen (CPu) and thalamus was demonstrated as diffuse and broad bands by Western blot with a polyclonal antibody against a C-terminal peptide of MOR, which were absent in the cerebellum and brains of MOR-knockout mice. The electrophoretic mobility of MOR differed in the two brain regions with median relative molecular masses (Mr’s) of 75 kDa (CPu) vs. 66 kDa (thalamus) for the rat, and 74 kDa (CPu) vs. 63 kDa (thalamus) for the mouse, which was due to its differential N-glycosylation. Rat MOR in CPu was found mainly associated with low-density cholesterol- and ganglioside M1 (GM1)-enriched membrane subdomains (lipid rafts), while the MOR in the thalamus was present in rafts and non-rafts without preference. Cholesterol reduction by methyl-β-cyclodextrin decreased DAGMO-induced [35S]GTPγS binding in rat CPu membranes to a greater extent than in the thalamus membranes.

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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