Dopamine receptors belong to the family of G protein-coupled receptors. On the basis of the homology between these receptors, three different dopamine receptors (D1, D2, D3) have been cloned. Dopamine receptors are primary targets for drugs used in the treatment of psychomotor disorders such as Parkinson's disease and schizophrenia. In the management of socially withdrawn and treatment-resistant schizophrenics, clozapine is one of the most favoured antipsychotics because it does not cause tardive dyskinesia. Clozapine, however, has dissociation constants for binding to D2 and D3 that are 4 to 30 times the therapeutic free concentration of clozapine in plasma water. This observation suggests the existence of other types of dopamine receptors which are more sensitive to clozapine. Here we report the cloning of a gene that encodes such a receptor (D4). The D4 receptor gene has high homology to the human dopamine D2 and D3 receptor genes. The pharmacological characteristics of this receptor resembles that of the D2 and D3 receptors, but its affinity for clozapine is one order of magnitude higher. Recognition and characterization of this clozapine neuroleptic site may prove useful in the design of new types of drugs.

Five or more dopine receptor genes are expressd in brain. However, the phacological similarities of the encoded DI-Ds receptors have hindered studies of the localizion and functions of the subtypes. To better understand the roles of the individual receptors, antibodies were raised apinst recombinant D1 and D2 proteins and were shown to bind to the receptor subtpes spefically in Western blot and immunoprecipitation studies. Each antibody reacted selectively with the r ive receptor protein expressed both in cells transfected with the cDNAs and in brain. By immunocytochemistry, D1 and D2 had similr reonal distibutions in rat, monkey, and human brain, with the most intense saning in sriatum, olfactory bulb, and substantia nigra. Within each region, however, the precise distributions ofeach subtype were dstinct and often complementary. Di and D2 were differentidy enriched in striatal patch and matrix compwrtments, in selective layers of the olfactory bulb, and in either substantia nigra pars compacta or reticulata. Electron microscopy demonstrated that D1 and D2 also had highly selective subcellular distributions. In the rat nestiatum, the majority of D1 and D2 immunoreactivity was localized in postsynaptic sites in subsets of spiny dendrites and spine heads in rat nosriatum. Presynaptic D1 and D2 receptors were also observed, indicating both ubtypes may regulate neurotransmitter rebase. DI was also present in axon terminals in the substantia nigra. These results provide a morphologkal substrate for understanding the preand postsynaptic functions of the genetically defined DI and D2 receptors in discrete neuronal circuits in mammalin brain.A family of at least five dopamine receptor genes, D1-D5, has been identified in both rodents and humans (1,2 1047-1339 (13) encoding the C-terminal 97 aa] and D2i3 [nt 661-1020 (14) encoding 120 aa of the i3 loop from the D2s splice variant] were subcloned into pGEX-2T, expressed as soluble fusion proteins in bacteria, and affinity-purified as described (12,15). The recombinant plasmids were confirmed by sequence analysis and encode a 27.5-kDa polypeptide fragment of glutathione S-transferase (GST) fused to the receptor polypeptide (Dic-GST and D%-GST). High yields (10-25 mg/ liter) ofpurified DIC-GST and D2v-GST fusion proteins were obtained.Antisera. Two female New Zealand White rabbits were immunized with each fusion protein. Animals received 100 jg of affinity-purified fusion protein in Freund's adjuvant followed by secondary injections at 3 weeks with the same dose, and then monthly booster injections with 25 ,ug of protein.Antibodies were affinity-purified on the respective purified fusion proteins conjugated to Affi-Gel (Bio-Rad) as described (12). Sera were preadsorbed with GST and bacterial lysates prior to affinity purification to remove antibodies reactive with the nondopamine receptor portion ofthe fusion proteins. The two antisera for each receptor yielded similar results.Immunoblot Analysis. SDS/PAGE was used to fractionate total protein from bacteria...

Dopamine D1-like receptors, composed of D1 and D5 receptors, have been documented to modulate glutamate-mediated fast excitatory synaptic neurotransmission. Here, we report that dopamine D1 receptors modulate NMDA glutamate receptor-mediated functions through direct protein-protein interactions. Two regions in the D1 receptor carboxyl tail can directly and selectively couple to NMDA glutamate receptor subunits NR1-1a and NR2A. While one interaction is involved in the inhibition of NMDA receptor-gated currents, the other is implicated in the attenuation of NMDA receptor-mediated excitotoxicity through a PI-3 kinase-dependent pathway.

Dopamine receptors belong to a superfamily of receptors that exert their biological effects through guanine nucleotide-binding (G) proteins. Two main dopamine receptor subtypes have been identified, D1 and D2, which differ in their pharmacological and biochemical characteristics. D1 stimulates adenylyl cyclase activity, whereas D2 inhibits it. Both receptors are primary targets for drugs used to treat many psychomotor diseases, including Parkinson's disease and schizophrenia. Whereas the dopamine D1 receptor has been cloned, biochemical and behavioural data indicate that dopamine D1-like receptors exist which either are not linked to adenylyl cyclase or display different pharmacological activities. We report here the cloning of a gene encoding a 477-amino-acid protein with strong homology to the cloned D1 receptor. The receptor, called D5, binds drugs with a pharmacological profile similar to that of the cloned D1 receptor, but displays a 10-fold higher affinity for the endogenous agonist, dopamine. As with D1, the dopamine D5 receptor stimulates adenylyl cyclase activity. Northern blot and in situ hybridization analyses reveal that the receptor is neuron-specific, localized primarily within limbic regions of the brain; no messenger RNA was detected in kidney, liver, heart or parathyroid gland. The existence of a dopamine D1-like receptor with these characteristics had not been predicted and may represent an alternative pathway for dopamine-mediated events and regulation of D2 receptor activity.

Mutations in alpha-synuclein, a protein highly enriched in presynaptic terminals, have been implicated in the expression of familial forms of Parkinson's disease (PD) whereas native alpha-synuclein is a major component of intraneuronal inclusion bodies characteristic of PD and other neurodegenerative disorders. Although overexpression of human alpha-synuclein induces dopaminergic nerve terminal degeneration, the molecular mechanism by which alpha-synuclein contributes to the degeneration of these pathways remains enigmatic. We report here that alpha-synuclein complexes with the presynaptic human dopamine transporter (hDAT) in both neurons and cotransfected cells through the direct binding of the non-A beta amyloid component of alpha-synuclein to the carboxyl-terminal tail of the hDAT. alpha-Synuclein--hDAT complex formation facilitates the membrane clustering of the DAT, thereby accelerating cellular dopamine uptake and dopamine-induced cellular apoptosis. Since the selective vulnerability of dopaminergic neurons in PD has been ascribed in part to oxidative stress as a result of the cellular overaccumulation of dopamine or dopamine-like molecules by the presynaptic DAT, these data provide mechanistic insight into the mode by which the activity of these two proteins may give rise to this process.