Rat brain cannabinoid receptors are N-linked glycosylated proteins

To study the N-linked glycosylation properties of the CB1 receptor, rat brain membranes were treated with exo- and endoglycosidases. For visualizing CB1 receptors, an antipeptide antibody was raised against the N-terminal 14 amino acids and used to specifically detect the protein by Western blotting. We found that the apparent molecular weight of mature CB1 receptors was 64 kDa. Treatment of membranes with endoglycosidase F shifted the 64 kDa band to the 59 kDa and 53 kDa bands. The latter is consistent with the calculated molecular weight of deglycosylated CB1 receptors. Treatment of membranes with endoglycosidase H and alpha-mannosidase partially shifted the 64 kDa band to 53 kDa band, indicating a portion of the oligosaccharides was of the high mannose type. These data confirmed that the CB1 receptors in brain are N-linked glycoproteins with heterogeneous carbohydrate composition. Among three potential N-linked glycosylation sites on the N-terminus of the CB1 receptor, only two sites are actually glycosylated.