Roles of protein kinase C and actin-binding protein 280 in the regulation of intracellular trafficking of dopamine D₃ receptor

D₃ dopamine receptor (D₃R) is expressed mainly in parts of the brain that control the emotional behaviors. It is believed that the improper regulation of D₃R is involved in the etiology of schizophrenia. Desensitization of D₃R is weakly associated with G protein-coupled receptor kinase (GRK)/β-arrestin-directed internalization. This suggests that there might be an alternative pathway that regulates D ₃R signaling. This report shows that D₃R undergoes robust protein kinase C (PKC)-dependent sequestration that is accompanied by receptor phosphorylation and the desensitization of signaling. PKC-dependent D ₃R sequestration, which was enhanced by PKC-β or -δ, was dynamin dependent but independent of GRK, β-arrestin, or caveolin 1. Site-directed mutagenesis of all possible phosphorylation sites within the intracellular loops of D₃R identified serine residues at positions 229 and 257 as the critical amino acids responsible for phorbol-12-myristate-13- acetate (PMA)-induced D₃R phosphorylation, sequestration, and desensitization. In addition, the LxxY endocytosis motif, which is located between residues 252 and 255, was found to play accommodating roles for PMA-induced D₃R sequestration. A continuous interaction with the actin-binding protein 280 (filamin A), which was previously known to interact with D₃R, is required for PMA-induced D₃R sequestration. In conclusion, the PKC-dependent but GRK-/β-arrestin-independent phosphorylation of D₃R is the main pathway responsible for the sequestration and desensitization of D₃R. Filamin A is essential for both the efficient signaling and sequestration of D₃R.