The dopamine D3 receptor interacts with itself and the truncated D3 splice variant d3nf: D3-D3nf interaction causes mislocalization of D3 receptors.

We have generated a stable cell line expressing FLAG epitope-tagged D3 dopamine receptors and used this cell line to study D3 receptor-protein interactions. To analyze protein interactions, we separately introduced into the stable cell line either D3 receptors carrying an hemagglutinin (HA) epitope tag, or an HA-tagged version of the D3 receptor splice variant D3nf. A combination of confocal laser microscopy and coimmunoprecipitation was used to assay the formation and expression pattern of D3-D3 homodimers or D3-D3nf heterodimers. When coexpressed in HEK 293 cells, FLAG- and HA-tagged D3 receptors exhibited a similar plasma membrane distribution. Using an HA epitope tag-specific antibody, we coimmunoprecipitated HA- and FLAG-tagged D3 receptors, suggesting that D3 receptors are capable of forming homodimers. Epitope-tagged D3nf polypeptides exhibited a markedly different cellular distribution than D3 receptors. When expressed in HEK 293 cells, either alone or in combination with FLAG-tagged D3 receptors, D3nf exhibited a punctate perinuclear distribution. When D3nf was introduced into the stable D3-expressing cell line, D3 receptors were no longer visualized at the plasma membrane. Instead, D3 and D3nf showed a similar, predominantly cytosolic, localization. Using the HA-specific antibody, we were able to coimmunoprecipitate D3 and D3nf polypeptides from transfected cells. These data suggest the existence of physical interaction between D3 and D3nf. This interaction appears to result in the mislocalization of D3 receptors from the plasma membrane to an intracellular compartment, a finding that could be of significance in the etiology of schizophrenia.

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.