Comparative pharmacological and functional analysis of the human dopamine D4.2 and D4.10 receptor variants.

The human dopamine D4 receptor is a D2-like receptor which is a target for most common neuroleptics. Previous investigations have shown that this receptor displays a large polymorphic variation in the third intracellular loop involving a variable number of direct imperfect tandem repeats (VNTR) of 16 amino acids. The shortest and longest repeat variants reported to date contain two and 10 repeat units (D4.2 and D4.10). No major pharmacological differences have been reported for the most common variants of this receptor (D4.2, D4.4 and D4.7), although the D4.7 was reported by us to display a slightly lower potency for dopamine in functional assays. Direct pharmacological and functional comparison of the longest and shortest variants in this study suggest no major discrepancies in pharmacological or functional profile between both receptors. Both receptors display, on average, a 15-fold and 90-fold lower potency for epinephrine and norepinephrine, respectively, compared with dopamine. We observed small increases in functional potency and affinity for dopamine and quinpirole at the D4.10 receptor variant compared with the D4.2 receptor. Our data indicate that there is no direct relationship between the length of the polymorphism and changes in pharmacology or functional activity. These findings are a suitable caution against the arbitrary pooling of D4 receptor VNTR genotypes in genetic studies, based on length.

Promoter-independent regulation of cell-specific dopamine receptor expression.

Here we describe the construction of recombinant adenoviruses expressing dopamine D2 and D4 receptors, and their ability to mediate high levels of heterologous expression in a variety of cell types in vitro and in vivo for at least 7 days post infection. These experiments demonstrated that maximum receptor expression is achieved generally within 24 h and remains constant thereafter. Maximum expression levels were highly variable between cell lines and dependent on infection efficiency and promoter strength. Correction for these two variables revealed differences in relative expression levels between cell lines varying by two orders of magnitude. Our results indicate that in addition to gene transcription, post-transcriptional mechanisms play a dominant role in determining dopamine receptor levels in this system.

Dopamine D2-like sites in schizophrenia, but not in Alzheimer's, Huntington's, or control brains, for [3H]benzquinoline.

Although the basis of schizophrenia is not known, evidence indicates a possible overactivity of dopamine pathways. In order to detect any new dopamine receptor-like sites which may be altered in schizophrenia, the present study used a new radioligand, a [3H]benzo[g]quinoline. The receptors were labelled by this ligand in the presence of other drugs to block the known dopamine D1, D2, D3, or D5 receptors (no D4-selective ligands are available to block D4). Using this method, we found that schizophrenia brain striata had elevated levels of a D2-like site not detected in control human postmortem brains or in Alzheimer's, Huntington's, or Parkinson's disease brains. The ligand acted as an agonist at this D2-like site, because binding was abolished by guanine nucleotide. The binding of the ligand to the D4 receptor, however, was not sensitive to guanine nucleotide. The site differed from D2 itself, because S- and R-sulpiride were equally potent at the D2-like site. The D2-like sites were present in rat and mouse brain but were absent in brain slices from transgenic mice where D2 had been knocked out. The abundance of the receptor was not related to premortem use of antipsychotic drugs. Future research should examine the biochemical differences between the D2 dopamine receptor and these D2-like sites in schizophrenia.

Cryptic initiation at the human D4 receptor reveals a functional role for the amino terminus.

It was found that deletion of the initiator methionine of the D4 receptor results in the use of a cryptic initiation site in the putative first transmembrane region. We made use of this observation to investigate the role of the amino terminus of the D4 receptor. In vitro transcription and translation of D4.4 and a D4.4 deleted for the initiation codon (D4.4 delta NH2) resulted in the formation of protein products with a molecular mass of about 44 and 40.5 kDa, respectively. The molecular mass of 40.5 kDa suggests initiation in the putative first transmembrane region. Transient expression of various deletion mutants indicated that this receptor form can be expressed at up to 70% of the D4.4 control levels and provided support for the existence for an alternative translation initiation site in the first transmembrane domain, most likely at nucleotide +112 (the initiator methionine codon is designated as +1). The D4.4 delta NH2 mutant was stably expressed in CHO cells. Pharmacological analysis demonstrated no major differences in antagonist binding with the regular D4.4 receptor, while dopamine and quinpirole binding affinities were about 5-fold decreased. The half-maximal level (EC50) for blocking forskolin-stimulated cAMP levels by dopamine was about 10-fold lower as compared to D4.4. Furthermore, the functional efficacy is decreased by about 40%. These data suggest that the amino-terminal domain is not essential for proper expression, but does interfere with the functional activity of the receptor, possibly through stabilization of the active state. To our knowledge this is the first demonstration that the amino terminus of a dopamine receptor is involved in signal transduction.

Schizophrenia: elevation of dopamine D4-like sites, using [3H]nemonapride and [125I]epidepride.

We here report a three-fold elevation of dopamine D4-like sites in schizophrenia, using [3H]nemonapride to measure dopamine D2 and D3 receptors and D4-like sites, and using [125I]epidepride to measure D2 and D3 sites in ten control and nine schizophrenia post-mortem brain putamen tissues. This result differs from a recent report which did not detect significant D4-like sites in control or schizophrenia putamen (Reynolds and Mason, 1995, Eur. J. Pharmacol. 281, R5). The present finding agrees with other reports wherein an elevation in D4-like sites was found in schizophrenia, using [3H]nemonapride for D2, D3 and D4-like sites, but [3H]raclopride for D2 and D3 sites. The nature of these D4-like sites is not known.

Functional characterization of the human dopamine D4.2 receptor using vaccinia virus as an expression system.

Recombinant vaccinia viruses harboring the human dopamine D4 receptor cDNA containing two 48 base pair-repeats (D4.2) or the rat dopamine D2 short (D2s) receptor cDNA were used to infect rat-1 fibroblasts. Heterologous expression of both dopamine receptors was demonstrated in binding assays. The affinity constants of these receptors were consistent with values previously reported, including D4.2's higher affinity for the antipsychotic clozapine and raclopride's selectivity for D2 receptors. In the presence of 200 microM 5'-guanylyl-imidodiphosphate (Gpp[NH]p) both receptors exhibited reduced affinities for dopamine. Furthermore, when rat-1 cells were infected with the D2s or the D4.2 recombinant vaccinia viruses and exposed to dopamine agonists, the inhibition of adenylyl cyclase activity was prevented in pertussis toxin-treated cells. This study demonstrates the utility of recombinant receptor-vaccinia viruses in studies of expression, pharmacology and functional coupling of inhibitory G protein-coupled receptors.

Long-term haloperidol elevates dopamine D4 receptors by 2-fold in rats.

Chronic treatment of rats with neuroleptics results in elevated numbers of dopamine D2-like receptors. The present study was done to determine whether neuroleptics altered the density of one type of dopamine D2-like receptors, namely the dopamine D4 receptor. We here describe the effect of a one-month treatment with haloperidol on dopamine D4 receptor mRNA and protein levels in rat striatum. Endogenous levels of dopamine D4 receptor mRNA in rat striatum are very low and, therefore, reverse transcription and subsequent amplification were used for quantification. Dopamine D4 receptor density was, because of the absence of a dopamine D4 receptor specific [3H]ligand, determined by the difference between the number of binding sites for [3H]nemonapride and [3H]raclopride. Scatchard analysis of [3H]nemonapride and [3H]raclopride binding show that treatment for one month with halperidol elevates the density of dopamine D4 receptors in rat striatum by approximately 2-fold, whereas dopamine D2 and D3 receptors together show a 19% higher receptor density. Dopamine D4 receptor mRNA was also approximately increased by 2-fold.

Binding of 5H-dibenzo[a,d]cycloheptene and dibenz[b,f]oxepin analogues of clozapine to dopamine and serotonin receptors.

Series of 5,11-dicarbo- and 11-carbo-5-oxy-10-(1-alkyl-1,2,3,6-tetrahydro-4 pyridinyl) analogues and a 11-carbo-5-oxy-10-(1-methyl-4-piperidinyl) analogue of the atypical antipsychotic agent clozapine were prepared and tested for binding to the dopamine D-2L and D-4 and serotonin S-2A and S-2C receptors. Some of these analogues were found to have dopamine D-2L and D-4 and serotonin S-2A and S-2C receptor binding activities as high as or higher than those of clozapine, indicating that neither the diazepine structure nor the piperazine ring present in clozapine is essential for high antidopamine activity and or for high dopamine D-4 selectivity (Ki for the dopamine D-2L receptor/Ki for the dopamine D-4 receptor). Increasing in the effective size of the alkyl substituent at the tertiary amine nitrogen atom in the 1,2,3,6-tetrahydro-4-pyridinyl moiety in the 5H-dibenzo[a,d]cycloheptene series reduces the affinity for the dopamine D-4 receptor, but in the dibenz[b,f]oxepin series, no significant change in binding affinity to the dopamine D-4 receptor was observed. Equal or slightly higher affinity for the serotonin S-2A and S-2C receptors was observed for the 10-(1-ethyl-1,2,3,6-tetrahydro-4- pyridinyl) analogues in both series, but for the 10-[1,2,3,6-tetrahydro-1-(2-propenyl)-4- pyridinyl] analogues, any favourable steric factor is overshadowed by an unfavorable electronic effect as a result of change in the basicity of the tertiary amino group in the pyridinyl moiety. Replacement of three of the four nitrogen atoms in clozapine with three carbon or two carbon atoms and an oxygen atom and removal of the chlorine atoms gives 10-(1,2,3,6-tetrahydro-1- methyl-4-pyridinyl)dibenzo[a,d]cycloheptene and 10-(1-methyl-4-piperidinyl)dibenz[b,f]oxepin, each having twice the binding activity to the dopamine D-4 receptor as does clozapine and a dopamine D-4 selectivity equal to that of clozapine.

Binding of 5H-dibenzo[b,e][1,4]diazepine and chiral 5H-dibenzo[a,d]cycloheptene analogues of clozapine to dopamine and serotonin receptors.

5H-Dibenzo[b,e][1,4]diazepine, dibenz[b,f]oxepin, and 5H-dibenzo[a,d]cycloheptene analogues of clozapine [8-chloro-11-(4-methylpiperazino)-5H- dibenzo[b,e][1,4]diazepine] were evaluated for their binding affinity to dopamine D-1, D-2, and D-4 and serotonin S-2A (5-HT2A), S-2C (5-HT2C) and S-3 (5-HT3) receptors. The diazepine analogues display selective binding to the dopamine D-4 and serotonin S-2A receptors similar to that of clozapine, but none has a dopamine D-4 selectivity (Ki for the dopamine D-2A receptor/Ki for the dopamine D-4 receptor) greater than that of clozapine. All of the oxepin analogues also show substantial binding to the dopamine D-4 and serotonin S-2A receptors with 10-(4-methylpiperazino)dibenz[b,f]oxepin having a dopamine D-4 selectivity greater than that of clozapine. Some of the 5H-dibenzo-[a,d]cycloheptene analogues also show strong binding to both the dopamine D-4 and serotonin S-2A receptors, 5-methyl-10-(4-methylpiperazino)-5H-dibenzo[a,d]cycloheptene having a dopamine D-4 selectivity of 7.8 as compared to 10 for clozapine but a serotonin S-2A selectivity (Ki for the dopamine D-2 receptor/Ki for the serotonin S-2A receptor) of 2.0 as compared to 28 for clozapine. The serotonin S-2A selectivity of 2-chloro-10-(4-methylpiperazino)-5H-dibenzo[a,d]-cycloheptene++ + is 200. As an extension of these studies, chiral 5-substitute 10-(1,2,3,6-tetrahydro-1-methyl-4-pyridinyl)-5H-dibenzo[a,d]cyclohept ene analogues show a substantial enantiospecificity toward dopamine and serotonin receptor subtypes, (R)-(-)-5-methyl compound having a 2-fold higher dopamine D-4 selectivity than its (S)-(+) enantiomer as the result of enhanced binding to the dopamine D-4 receptor rather than diminished binding to the dopamine D-2 receptor. (pRa,pSb)-(+)-5-(2-Propylidene)-10-(1,2,3,6-tetrahydro-1-met hyl- 4-pyridinyl)-5H-dibenzo[a,d]cycloheptene is 17 times more active in binding to the dopamine D-4 receptor than is its pSa,pRb enantiomer while being only 1.5 times more active in binding to the dopamine D-2 receptor.

Dopamine D4 receptor repeat: analysis of different native and mutant forms of the human and rat genes.

Recent molecular characterization of the human D4 gene has revealed the existence of various polymorphic forms of this receptor. These variations are found in the putative third cytoplasmic loop region and encode a variable number of repeats of 16 amino acids in length. In the present study we have compared the pharmacological binding profiles of seven different polymorphic variants of the human D4 receptor, the rat D4 receptor, and two different human D4 receptor mutants that were deleted in the repeat sequence. For this purpose we cloned the rat D4 receptor gene and compared its gene structure and its pharmacological binding profile with those of the D4.4 and D4.7 genes. The rat and human D4 genes display a high degree of sequence similarity, especially in the coding regions. An Alu repeat sequence was identified in the first intron of the human D4 gene but is not present in the rat D4 gene. Furthermore, using the polymerase chain reaction we cloned 3-, 5-, 6-, and 9-fold repeat sequences. These cloned repeat sequences were used for the reconstruction of full length cDNAs encoding D4.3, D4.5, D4.6, and D4.9, respectively. These novel forms of the human D4 receptor, as well as the previously cloned D4.2, D4.4, and D4.7 forms, were transiently expressed in COS-7 cells. All of the different forms of the human and rat D4 receptors and repeat deletion mutants displayed similar binding profiles for all ligands tested, although small differences were observed. The affinity for dopamine could be decreased by guanosine-5'-(beta, gamma-imido)triphosphate with the different forms of the D4 receptor, including the two receptor mutants that were deleted in the repeat sequence. These data suggest that the polymorphic repeat sequence has little influence on D4 binding profiles and might not be essential for G protein interaction.

Dopamine D4 receptors elevated in schizophrenia.

Although the biological basis of schizophrenia is not known, possible causes include genetic defects, viruses, amines, brain structure and metabolism, neuroreceptors, and G proteins. The hypothesis of dopamine overactivity in schizophrenia is based on the fact that neuroleptics block dopamine D2 receptors in direct relation to their clinical antipsychotic potencies. Moreover, dopamine D2 or D2-like receptors are elevated in postmortem schizophrenia brain tissue. This elevation, however, is only found in vivo using [11C]methylspiperone but not [11C]raclopride. The dopamine D4 receptor gene has not yet been excluded in schizophrenia because the 21 gene variants of D4 have not yet been tested. Because the link between D1 and D2 receptors is reduced in schizophrenia tissue, we tested whether one component of this link was sensitive to guanine nucleotide. We report here that the binding of [3H]raclopride to D2 receptors in schizophrenia was not sensitive to guanine nucleotide. This finding permitted analysis of data on the binding of [3H]emonapride to the D2, D3 and D4 receptors. We conclude that the combined density of D2 and D3 receptors (labelled by [3H]raclopride) is increased by only 10% in schizophrenia brain, as found by Farde et al., but that it is the density of dopamine D4 receptors which is sixfold elevated in schizophrenia. These findings resolve the apparent discrepancy, mentioned above, wherein the density of [11C]methylspiperone-labelled sites (D2, D3 and D4), but not that of [11C]raclopride-labelled sites (D2 and D3), was found elevated in the schizophrenia striatum.

Low density of dopamine D4 receptors in Parkinson's, schizophrenia, and control brain striata.

The purpose of this study was to determine whether dopamine D4 receptors could be detected in the human brain striatum by means of an indirect ligand-binding method, because no dopamine D4 receptor-selective ligand presently exists. The antipsychotic clozapine is more selective for the dopamine D4 receptor than for other dopamine receptors. Although most antipsychotic drugs act in the striatum to elicit Parkinson-like side-effects, clozapine is atypical in that it does not produce Parkinsonism. To understand this atypical action of clozapine, it would be helpful to know whether the presumed target for clozapine, the dopamine D4 receptor, is or is not present in the human striatum. We measured dopamine D4 receptors indirectly, using [3H]emonapride and [3H]raclopride. Emonapride has a high affinity (K = 90 pM) for the dopamine D4 receptor, while raclopride has a very low affinity for this receptor (K = 240 nM); thus, any difference in the densities of these two [3H]ligands (in the absence of dopamine) could be attributed to the presence of dopamine D4 receptors. Since the binding of [3H]raclopride is sensitive to endogenous dopamine, we used Parkinson-diseased tissue which has little dopamine. We found that the densities of the two ligands were identical in Parkinson striata, indicating a low density (< 1 pmol/g) for dopamine D4 receptors in the human striatum. This low or undetectable density of dopamine D4 receptors in the striatum is consistent with other data indicating that clozapine does not have its major action in the human striatum.(ABSTRACT TRUNCATED AT 250 WORDS)

Serotonin receptor cDNA cloned from Lymnaea stagnalis.

Serotonin (5-HT) is a major neurotransmitter that influences various behaviors, neuronal plasticity, learning, and memory in molluscs. Although the physiology of 5-HT transmission in molluscs is well studied, little is known about the pharmacology and diversity of the 5-HT receptor system. Based on the high homology of genes coding for guanine nucleotide-binding protein (G protein)-coupled receptors, we have cloned a gene for the Lymnaea stagnalis 5-HT (5HTlym) receptor. The putative receptor protein, 509 amino acids long, has highest homology with the Drosophila 5-HT receptors and mammalian 5HT1 receptors. As revealed by RNA blot-hybridization analysis, two mRNA species of 2.3 and 3.2 kb are detected in the central nervous system of Lymnaea. Transient expression of 5HTlym in COS-7 cells showed saturable [3H]lysergic acid diethylamide binding with an estimated dissociation constant of 0.9 nM. The 5HTlym receptor exhibited a mixed 5HT-like pharmacology that cannot be precisely categorized with existing mammalian classification nomenclature. However, the 5HTlym receptor does display some characteristics that have been attributed to the putative mammalian vascular 5HT1-like receptor.

The cloned dopamine D2 receptor reveals different densities for dopamine receptor antagonist ligands. Implications for human brain positron emission tomography.

Since [3H]emonapride ([3H]YM-09151-2), a benzamide neuroleptic, consistently detects more dopamine D2 receptors than [3H]spiperone in the same tissue, we tested whether this property was inherent in the cloned dopamine D2 receptor. We found that the density of dopamine D2 receptors labelled by [3H]emonapride was 1.5-fold to 2-fold (mean of 1.8-fold) higher than the density of dopamine D2 receptors labelled by [3H]spiperone in cells expressing cloned dopamine D2 receptors (either the short form (from rat) or the long form (from human)), matching similar findings in anterior pituitary tissue (rat or pig) or in post-mortem human caudate nucleus tissue. The situation was similar for another benzamide, [3H]raclopride, which revealed 1.3-fold to 1.8-fold (mean of 1.5-fold) more binding sites than that for [3H]spiperone in cell membranes containing cloned dopamine D2 receptors. The apparently different dopamine D2 receptor densities revealed by these two types of 3H-ligands (i.e. [3H]spiperone and the [3H]benzamides), therefore, arise from an inherent property of the dopamine D2 receptor protein. These findings for the cloned dopamine D2 receptor, therefore, partly explain the higher dopamine D2 receptor density measured in human brain (by positron emission tomography) when using radioactive raclopride compared to results using radioactive methylspiperone.(ABSTRACT TRUNCATED AT 250 WORDS)

Multiple dopamine D4 receptor variants in the human population.

The dopamine D4 receptor structurally and pharmacologically resembles the dopamine D2 and D3 receptors. Clozapine, an atypical antipsychotic that is relatively free of the adverse effects of drug-induced parkinsonism and tardive dyskinesia, binds to the D4 receptor with an affinity 10 times higher than to the D2 and D3 receptors. This may explain clozapine's atypical properties. Here we report the existence of at least three polymorphic variations in the coding sequence of the human D4 receptor. A 48-base-pair sequence in the putative third cytoplasmic loop of this receptor exists either as a direct-repeat sequence (D4.2), as a fourfold repeat (D4.4) or as a sevenfold repeat (D4.7). Two more variant alleles were detected in humans. Expression of the complementary DNA for the three cloned receptor variants showed different properties for the long form (D4.7) and the shorter forms (D4.2, D4.4) with respect to clozapine and spiperone binding. To our knowledge, this is the first report of a receptor in the catecholamine receptor family that displays polymorphic variation in the human population. Such variation among humans may underlie individual differences in susceptibility to neuropsychiatric disease and in responsiveness to antipsychotic medication.

(+/-)-3-allyl-7-halo-8-hydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepines as selective high affinity D1 dopamine receptor antagonists: synthesis and structure-activity relationship.

Substituted 1-phenyl-3-benzazepines form a class of compounds possessing potent and selective affinity for the D1 DA receptor. 7,8-Dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine (SKF 38393) and its 6-halo analogues are potent and selective D1 receptor agonists. Recently, the 3-allyl derivatives of SKF 38393 and its analogues were described as selective D1 agonists with higher D1 efficacy and CNS potency. In order to extend these results to compounds in the 7-halo-8-hydroxy-substituted antagonist series, we have synthesized and pharmacologically characterized 3-allyl analogues of 7-substituted (Cl, Br, H) 8-hydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepines. These 3-allyl derivatives were compared with their 3-methyl and 3-unsubstituted analogues in terms of their D1 receptor affinity and selectivity. The results have been used to generate structure-affinity relationships. The D1 receptor affinity, for 3-substitution, is found to be in the order: methyl greater than allyl greater than H. For 7-substitution, the affinity is in the order: Cl = Br greater than H. The 3-allyl compounds show affinity close to that of the parent (3-methyl) compounds while exhibiting a slightly diminished D1 selectivity. However, the greater lipophilicity of the 3-allyl compounds may enable them to cross the blood-brain barrier more readily and thereby exhibit higher in vivo CNS potency. Thus 3-allylbenzazepines have potential as high affinity selective D1 antagonists.

(+/-)-3-Allyl-6-bromo-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3- benzazepin, a new high-affinity D1 dopamine receptor ligand: synthesis and structure-activity relationship.

The 7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepines form a series of compounds having a high affinity at the D1 dopamine receptor. The 6-chloro derivative has been previously shown to have enhanced affinity, selectivity, and agonist activity. In an attempt to study the effect of substitution of a 6-bromo group in place of the 6-chloro, we have synthesized a series of compounds and evaluated them for their affinity for the D1 receptor. The results show that the 6-bromo derivatives have virtually identical affinities to their 6-chloro counterparts, a finding similar to that found in the D1 antagonist 7-halo-8-hydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine series. From the present work, 3-allyl-6-bromo-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepi ne (6-Br-APB) has been identified as a suitable candidate for further in vivo studies and resolution into its active and inactive enantiomers.

Cloning of the gene for a human dopamine D5 receptor with higher affinity for dopamine than D1.

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.

Cloning of the gene for a human dopamine D4 receptor with high affinity for the antipsychotic clozapine.

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.