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 surface expression of the D1 and D5 dopamine receptor subtypes. Treatment of transfected HEK 293 cells with tunicamycin, an inhibitor of N-linked oligosaccharide addition, was found to prevent localization of D5 receptors in the plasma membrane. In contrast, tunicamycin treatment had no effect on the plasma membrane localization of the D1 receptor. Polymerase chain reaction mutagenesis was used to generate a panel of D5 receptors containing mutations in the three predicted sites of N-linked glycosylation. Expression of mutant receptors indicated that glycosylation of residue N7 was the major determinant of D5 receptor plasma membrane localization. Mutation of a comparable site in the D1 receptor at position N5 had no effect on the delivery of the D1 receptor to the cell surface. Tunicamycin treatment during receptor biosynthesis, but not N-glycosidase F digestion of mature receptors, abrogated binding of the D5 receptor antagonist [(3)H]SCH23390, suggesting that while 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.
Karpa
K D
KD
Department of Pharmacology, Penn State College of Medicine, Milton S. Hershey Medical Center, Hershey, Pennsylvania 17033, USA. kjd136@psu.edu
Lidow
M S
MS
Pickering
M T
MT
Levenson
R
R
Bergson
C
C
D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism*; D002478:Cells, Cultured; D006031:Glycosylation; D006801:Humans; D008024:Ligands; D009154:Mutation; D009844:Oligosaccharides / Q000494:pharmacology; D017447:Receptors, Dopamine D1 / Q000235:genetics / Q000378:metabolism*; D050641:Receptors, Dopamine D5; D014162:Transfection; D014415:Tunicamycin / Q000494:pharmacology
C493605:DRD5 protein, human; D008024:Ligands; D009844:Oligosaccharides; D017447:Receptors, Dopamine D1; D014415:Tunicamycin; D050641:Receptors, Dopamine D5