The dopamine D3 receptor interacts with itself and the truncated D3 splicevariant D3nf: D3-D3nf interaction causes mislocalization of D3 receptors

We have generated a stable cell line expressing FLAG epitope-tagged D3 dopa mine receptors and used this cell line to study D3 receptor-protein interac tions. To analyze protein interactions, we separately introduced into the s table cell line either D3 receptors carrying an hemagglutinin (HA) epitope tag, or an HA-tagged version of the D3 receptor splice variant D3nf. A comb ination of confocal laser microscopy and coimmunoprecipitation was used to assay the formation and expression pattern of D3-D3 homodimers or D3-D3nf h eterodimers. When coexpressed in HEK 293 cells, FLAG- and HA-tagged D3 rece ptors exhibited a similar plasma membrane distribution. Using an HA epitope tag-specific antibody, we coimmunoprecipitated HA- and FLAG- tagged D3 rec eptors, suggesting that D3 receptors are capable of forming homodimers. Epi tope-tagged D3nf polypeptides exhibited a markedly different cellular distr ibution than D3 receptors. When expressed in HEK 293 cells, either alone or in combination with FLAG- tagged D3 receptors, D3nf exhibited a punctate p erinuclear distribution. When D3nf was introduced into the stable D3-expres sing cell line, D3 receptors were no longer visualized at the plasma membra ne. Instead, D3 and D3nf showed a similar, predominantly cytosolic, localiz ation. Using the HA- specific antibody, we were able to coimmunoprecipitate D3 and D3nf polypeptides from transfected cells. These data suggest the ex istence of physical interaction between D3 and D3nf. This interaction appea rs to result in the mislocalization of D3 receptors from the plasma membran e to an intracellular compartment, a finding that could be of significance in the etiology of schizophrenia.