N-linked glycosylation is required for plasma membrane localization of D5,but not D1, dopamine receptors in transfected mammalian cells

We have analyzed the role of N-linked glycosylation in functional cell surf ace expression of the D1 and D5 dopamine receptor subtypes. Treatment of tr ansfected HEK 293 cells with tunicamycin, an inhibitor of N-linked oligosac charide addition, was found to prevent localization of D5 receptors in the plasma membrane. In contrast, tunicamycin treatment had no effect on the pl asma membrane localization of the D1 receptor. Polymerase chain reaction mu tagenesis was used to generate a panel of D5 receptors containing mutations in the three predicted sites of N-linked glycosylation. Expression of muta nt receptors indicated that glycosylation of residue N7 was the major deter minant of D5 receptor plasma membrane localization. Mutation of a comparabl e site in the D1 receptor at position N5 had no effect on the delivery of t he D1 receptor to the cell surface. Tunicamycin treatment during receptor b iosynthesis, but not N-glycosidase F digestion of mature receptors, abrogat ed binding of the D5 receptor antagonist [H-3]SCH23390, suggesting that whi le oligosaccharide moieties play a key role in the cell surface expression of D5 receptors, they do not appear to contribute to the receptor's ligand binding properties. Together, our data indicate a differential requirement for N-linked glycosylation in functional cell surface expression of D1 and D5 dopamine receptors.