The dopamine transporter (DAT) regulates the clearance of dopamine (DA) released into the extracellular space and is an important site on which psychostimulants act to produce their effects. Here, we show that mitogen-activated protein kinase (MAPK) regulates the transport capacity and intracellular trafficking of DAT. Incubation of striatal synaptosomes or epitope-tagged human DAT (hDAT) human embryonic kidney (HEK) 293 cells with the MAPK kinase (MEK) inhibitors 1,4-diamino-2,3-dicyano-1,4-bis(o-aminophenylmercapto) butadiene and 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one decreased DA uptake in a concentration- and time-dependent manner. Kinetic studies revealed a decrease in the capacity of transport (Vmax) but no change in Km. Immunoblotting confirmed labeling of p42 and p44 MAPK in untreated striatal synaptosomes and HEK 293 cells, consistent with constitutive MAPK activation, and the inhibitors used decreased MAPK phosphorylation. Biotinylation and confocal imaging studies showed that MAPK inhibition promoted the clathrin-associated redistribution of hDAT from the plasma membrane to the cytosol. In contrast, transient transfection of hDAT-expressing cells with constitutively active MEK increased the Vmax of DA transport without altering Km. However, only a small increase in hDAT cell surface expression was seen. These data demonstrate an involvement of the MAPK cascade in regulating DAT transport capacity in striatum and that inhibition of this cascade decreases DAT cell surface expression in HEK 293 cells. Furthermore, they highlight the potential role of MAPK as a presynaptic mechanism that regulates DA signaling.

ABSTRACT,u opiate receptors recognize morphine with high affinity. A 2.1-kb rat brain cDNA whose predicted translation product displays 63% identity with recently described 8 and K opiate receptor sequences was identified through polymerase chain reaction and cDNA homology approaches. This cDNA recognizes a 10.

Radioligand binding and cDNA homology studies have suggested the existence of opiate receptors distinct from the recently-cloned p, S and K receptors. XORlS, a rat brain cDNA whose predicted translation product displays 67-72% homology with those encoded by ~1, 61 and ~1 opiate receptor cDNAs, was constructed from two partial cDNAs identified through cDNA homology approaches. A longer XORlL variant of this cDNA was also identified by polymerase chain reaction studies using genomic DNA and cDNA from brain and peripheral tissues. XORl mRNA is most highly expressed in hypothalamus. COS cell expression of both clones confers neither robust binding of opiate hgands nor reproducible opiate inhibition of forskolin-stimulated adenylate cyclase. These studies identify an orphan clone that helps to define features of the opiate receptor gene family, including apparent differential splicing and expression in peripheral tissues.