Activation of dopamine D 1 receptors is critical for the generation of glutamate-induced long-term potentiation at corticostriatal synapses. In this study, we report that, in striatal neurons, D 1 receptors are co-localized with N-methyl-D-aspartate (NMDA) receptors in the postsynaptic density and that they co-immunoprecipitate with NMDA receptor subunits from postsynaptic density preparations. Using modified bioluminescence resonance energy transfer, we demonstrate that D 1 and NMDA receptor clustering reflects the existence of direct interactions. The tagged D 1 receptor and NR1 subunit cotransfected in COS-7 cells generated a significant bioluminescence resonance energy transfer signal that was insensitive to agonist stimulation and that did not change in the presence of the NR2B subunit, suggesting that the D 1 receptor constitutively and selectively interacts with the NR1 subunit of the NMDA channel. Oligomerization with the NR1 subunit substantially modified D 1 receptor trafficking. In individually transfected HEK293 cells, NR1 was localized in the endoplasmic reticulum, whereas the D 1 receptor was targeted to the plasma membrane. In cotransfected cells, both the D 1 receptor and NR1 subunit were retained in cytoplasmic compartments. In the presence of the NR2B subunit, the NR1-D 1 receptor complex was translocated to the plasma membrane. These data suggest that D 1 and NMDA receptors are assembled within intracellular compartments as constitutive heteromeric complexes that are delivered to functional sites. Coexpression with NR1 and NR2B subunits also abolished agonist-induced D 1 receptor cytoplasmic sequestration, indicating that oligomerization with the NMDA receptor could represent a novel regulatory mechanism modulating D 1 receptor desensitization and cellular trafficking. Dopaminergic fibers originating in the substantia nigra and cortical glutamatergic neurons extensively interact in the striatum to drive the physiological functions of this structure from motor planning to reward seeking and procedural learning (1, 2). The critical importance of dopamine in this system is such that the degeneration of nigral dopaminergic neurons leads to the motor and cognitive deficits of Parkinson's disease (3).At the cellular level, nigral and cortical fibers converge on the medium spiny projection neurons (4), where dopamine D 1 -and D 2 -like receptors are coexpressed to high degree with glutamate NMDA 1 and non-NMDA receptor channels (5-8). From a functional point of view, it is well established that dopamine modulates the firing pattern of these neurons. In particular, there is evidence that dopamine, while attenuating the responses mediated by non-NMDA receptors, potentiates those associated with activation of NMDA receptors (2). The D 1 receptor appears to be involved in these interactions. In fact, activation of D 1 receptors in medium spiny neurons enhances NMDA-induced whole cell currents (2, 9) and is a critical requirement for the formation of NMDA-mediated long-term potentiation at corticostriatal...

Colocalization of dopamine D1 (D1R) and D3 receptors (D3R) in specific neuronal populations suggests that their functional cross-talk might involve direct interactions. Here we report that the D1R coimmunoprecipitates with the D3R from striatal protein preparations, suggesting that they are clustered together in this region. Using bioluminescence resonance energy transfer (BRET 2 ), we further suggest the existence of a physical interaction between D1R and D3R. Tagged D1R and D3R cotransfected in human embryonic kidney (HEK) 293 cells generated a significant BRET 2 signal that was insensitive to agonist stimulation, suggesting that they form a constitutive heterodimer. D1R and D3R regulate adenylyl cyclase (AC) in opposite ways. In HEK 293 cells coexpressing D1R and D3R, dopamine stimulated AC with higher potency and displaced [ 3 H]R-(ϩ)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SCH23390) binding with higher affinity than in cells expressing the D1R. In HEK 293 cells individually expressing D1R or D3R, agonist stimulation induces internalization of D1R but not of D3R. Heterodimerization with D3R abolishes agonist-induced D1R cytoplasmic sequestration induced by selective D1R agonists and enables internalization of the D1R/ D3R complex in response to the paired stimulation of both D1R and D3R. This mechanism involves ␤-arrestin binding because it was blocked by mutant ␤-arrestinV53D. These data suggest that as a result of dimerization, the D3R is switched to the desensitization mechanisms typical of the D1R. These data give a novel insight into how D1R and D3R may function in an integrated way, providing a molecular mechanism by which to converge D1R-and D3R-related dysfunctions. Dopamine (DA) controls various physiological functions, including locomotor activity, learning and memory, and motivation and reward; dopaminergic dysfunctions have been implicated in the development of Parkinson's disease, schizophrenia, and drug abuse. DA acts through five receptors, belonging to the G protein-coupled receptor (GPCR) family, that are divided into D1-like (D1 and D5) and D2-like (D2, D3, and D4) subtypes. Each receptor displays unique properties, including affinity for DA and specificity for G protein coupling and signaling and shows a peculiar neuronal distribution (Missale et al., 1998). The D1 receptor (D1R) is the most abundant and widespread DA receptor in the brain, where it is found at high density in both motor and limbic areas (Missale et al., 1998). The D3 receptor (D3R) is less abundant and exhibits a more restricted pattern of distribution with high concentrations in the ventral striatum, particularly in the shell of the nucleus accumbens and islands of Calleja (Sokoloff et al., 1990;Lévesque et al., 1992) and lower expression in other brain regions (Sokoloff et al., 1990;Lévesque et al., 1992;Schwartz et al., 1998). Both D1R and D3R have been implicated in the regulation of rewarding mechanisms and motivated behavior and in the modulation of emotional and cognitive p...

The dopamine transporter (DAT) is a presynaptic plasma membrane protein responsible for the termination of dopaminergic neurotransmission in the central nervous system. While most studies have focused on structure/function analysis, much less information is available regarding the assembly and the trafficking of this protein. To address this problem, we performed a mutational analysis of the DAT protein, combined with biochemical, immunological, and functional approaches. In mammalian cells co-expressing differentially tagged DAT molecules, HA-tagged DAT co-purified with 6His-tagged DAT demonstrating a physical interaction between transporter proteins. Evidence for the functional oligomerization of DAT was obtained using dominant-negative mutants of DAT. Two loss-of-function mutant transporters (Y335A and D79G) that were targeted to the cell surface inhibited wild-type DAT uptake activity without affecting the membrane targeting of the wildtype transporter. Moreover, non-functional amino and carboxyl termini-truncated mutants of DAT inhibited wild-type DAT function by interfering with the normal processing of the wild-type transporter to the cell membrane. Mutations in the leucine repeat of the second transmembrane domain of the transporter could eliminate the dominant-negative effect of all these mutants. In addition, a small fragment comprising the first two transmembrane domains of DAT inhibited wild-type transporter function but not when the leucine repeat motif was mutated. Taken together, our results suggest that the assembly of DAT monomers plays a critical role in the expression and function of the transporter. The dopamine transporter (DAT)1 belongs to a large family of Na ϩ /Cl Ϫ -dependent plasma membrane transporters that also includes the closely related norepinephrine and serotonin transporters (NET and SERT, respectively), and carriers for GABA, glycine, proline, taurine, and betaine. In the central nervous system, DAT mediates the re-uptake of released dopamine (DA) from the synaptic cleft back into the nerve terminal for subsequent storage and release. Pharmacological and genetic studies highlight the DAT-mediated re-uptake process as the main mechanism for the termination of dopamine neurotransmission (1). In addition, DAT represents the main target site for commonly abused drugs such as cocaine and amphetamine as well as some therapeutic agents used in the management of affective disorders (2). Hydrophobicity analysis of their deduced amino acid sequence reveals that Na ϩ /Cl Ϫ -dependent plasma membrane neurotransmitter transporters are proteins containing twelve transmembrane domains (TMs) with both the amino and the carboxyl termini located on the intracellular side of the membrane. This topological arrangement has been confirmed for several members of the family, including DAT (3). Since the molecular cloning of this transporter gene family, a great deal of information has been accumulated concerning the relationship between the structure and function of this class of proteins (4). Studies...