Gene encoding the rat dopamine D4 receptor

A gene, flanking 5&#8242; and 3&#8242; sequences and derived cDNA encoding a rat D4 dopamine receptor that is predominantly located in the cardiovascular and retinal systems is disclosed. The cDNA has been expressed in transfected mammalian cells and demonstrated to preferentially bind dopamine antagonists such as clozapine. The cDNA is useful as a probe for related D4 dopamine receptors. Expressed in appropriate cell lines, it is useful as an in vitro screen for drugs which specifically bind to the receptor. Drugs that specifically bind to the receptor are then screened using standard methodology in rats, mice or dogs, for the physiological effects. Amino acids deduced from the determination of cDNA can be used to generate either polyclonal or monoclonal antibodies which recognize the D4 receptor sequence but do not recognize D1, D2, D3 or D5 dopamenergic receptors, for use in immunocytochemical studies, and for identification and isolation via flow sorting of D4 expressing cell types. Antibodies could also be used to block or modify the effects of D4 agonists and/or antagonists. It is also demonstrated that selective stimulation or inhibition of some dopamine receptors, including D4, can be used to induce changes in the morphology of cells such as neurons.

BACKGROUND OF THE INVENTION

The present invention is generally in the area of dopamine receptors, and is specifically a gene encoding a dopamine D 4 receptor, its flanking 5 and 3 sequences, and its derived cDNA, and methods of use thereof in screening for compounds having selective effects on the cardiovasculature and retinal tissues through interactions with the dopamine D 4 receptor.

Dopamine is an important neurotransmitter in the central nervous system (CNS), where it is thought to be involved in a variety of functions including motor coordination, reproductive regulation, and generation of emotions. A distinct peripheral dopaminergic system is thought to exist, although it is less well characterized. CNS dopamine receptors have historically been divided into two major classes, D 1 and D 2 , which can be distinguished by pharmacological, functional, and physical characteristics (Kebabian and Calne, (1979) Multiple receptors for dopamine Nature 277:93-96; Hamblin et al., (1984) Interactions of agonists with D 2 dopamine receptors: evidence for a single receptor population existing in multiple agonist affinity-states in rat striatal membranes Biochem. Pharmacol. 33:877-887; Seeman et al., (1985) Conversion of dopamine receptors from high to low affinity for dopamine Biochem. Pharmacol. 34:151-154; Niznik, (1987) Dopamine receptors: molecular structure and function Mol. Cell. Endocrinol. 54:1-22). Peripheral dopamine receptors have been divided into DA1 and DA2 subgroups, which share some but not all pharmacological characteristics with their CNS counterparts (Goldberg and Kohli, (1987) Identification and characterization of dopamine receptors in the cardiovascular system Cardiologia 32:1603-1607; Kohli et al., (1989) Dopamine receptors in the stellate ganglion of the dog Eur. J. Pharmacol. 164:265-272; Brodde, (1990) Physiology and pharmacology of cardiovascular catecholamine receptors; implications for treatment of chronic heart failure Am. Heart J. 120:1565-1572).

Molecular cloning techniques have revealed a diversity of CNS receptor subtypes in each class. All are members of the G protein-coupled receptor gene superfamily and have seven potential transmembrane (Tm) spanning domains. In contrast to most members of the G-protein coupled receptor gene family, the D 2 -like genes have multiple exons separated by introns both in the coding and non-coding regions. Further diversity is generated by alternative splicing.

Prototypic D 2 ligand binding and signal transduction characteristics have been found for D 2 (Bunzow et al., (1988) Cloning and expression of a rat D 2 dopamine receptor cDNA Nature 336:783-787) and D 3 (Sokoloff et al., (1990) Molecular cloning and characterization of a novel dopamine receptor (D 3 ) as a target for neuroleptics Nature 347:146-151) receptors. The recently reported human D 4 receptor also has a D 2 -like pharmacological profile (Van Tol et al., (1991) Cloning of the gene for a human dopamine D 4 -receptor with high-affinity for the antipsychotic clozapine Nature 350-610-614). Two distinct D 1 receptors have also been cloned, called D 1 (Sunahara et al., (1990) Human dopamine D 1 receptor encoded by an intronless gene on chromosome 5 Nature 347:80-83; Zhou et al., (1990) Cloning and expression of human and rat D 1 dopamine receptors Nature 347:76-80; Monsma et al., (1990) Molecular cloning and expression of a D 1 dopamine receptor linked to adenylyl cyclase activation Proc. Natl. Acad. Sci. USA 87:6723-6727; Dearry et al., (1990) Molecular cloning and expression of the gene for a human D 1 dopamine receptor Nature 347:72-76) and D 5 (Sunahara et al., (1991) Cloning of the gene for a human dopamine D 5 receptor with higher affinity for dopamine than D 1 Nature 350:614-619). To date no peripheral dopamine receptor has been cloned, although it has been suggested that there is a low level of expression of D 3 in kidney (Sokoloff et al., 1990).

Van Tol et al. (1991) reported the isolation of a human D 4 receptor with a high affinity for the neuroleptic drug clozapine. Multiple variants of this dopamine receptor were also reported by Van Tol, et al., (1992) Nature 358, 149-154. These receptors were also the subject of PCT WO 92/10571 by State of Oregon. Although the function of these particular receptors was not identified, they are assumed to be important in binding drugs having anti-psychotic activity.

It is an object of the present invention to provide the gene, its flanking 5 and 3 sequences and the derived cDNA encoding another dopamine D 4 receptor present in rat cells.

It is a further object of the present invention to provide methods for expression and screening of compounds binding the new dopamine D 4 receptor.

It is another object of the present invention to provide a method for screening for compounds having cardiovascular activity and effects on retinal tissue which specifically bind to dopamine D 4 receptors.

It is still another object of the present invention to provide a means and method for modulation of the morphology of cells expressing D 4 receptors, and other dopamine receptors, by stimulation or inhibition of the receptors via exposure of the cells to specific compounds.

SUMMARY OF THE INVENTION

A gene, its 5 and 3 flanking sequences and the derived cDNA encoding a rat D 4 dopamine receptor that is predominantly located in the cardiovascular and retinal systems is disclosed. The gene has been expressed in transfected mammalian cells and demonstrated to preferentially bind dopamine antagonists such as clozapine.

The gene and/or cDNA is useful as a probe for related D 4 dopamine receptors. Expressed in appropriate cell lines, it is useful as an in vitro screen for drugs which specifically bind to the receptor. Drugs that specifically bind to the receptor are then screened using standard methodology in rats, mice or dogs, for the physiological effects. Antibodies to the protein are useful in immunocyto chemical studies, identification and isolation via flow sorting of D4 expressing cell types, and in blocking or modifying the effects of D4 agonists and/or antagonists.

Stimulation or inhibition of the D 4 receptor, D 2 receptor, or D 3 receptor, either in cells naturally expressing the receptor or which have been transfected with cDNAs or genes encoding anyone or more of several dopamine receptors, has been demonstrated to allow modification of the cell morphology. In one example, the number and extent of branching of neurites in cells transfected with dopamine receptors is increased significantly by exposure to compounds selectively binding to the receptors.

DETAILED DESCRIPTION OF THE INVENTION

Dopamine receptors have been implicated in a variety of neurological and neuropsychiatric disorders. The polymerase chain reaction and low stringency library screening were used to isolate a rat genomic clone encoding a new dopamine receptor. Sequence data and pharmacological analysis reveal this clone is the rat analog of the human D 4 receptor, which exhibits a high affinity for the antipsychotic drug clozapine. The mRNA for this receptor shows a restricted pattern of expression in the central nervous system. Significant levels of expression were found in the hypothalamus, thalamus, olfactory bulb, and frontal cortex. However, 20-fold higher levels of D 4 mRNA expression were observed in the cardiovascular system. High levels are also expressed in the photoreceptor layer of the retina. Stimulation of this receptor in the dark leads to a marked decrease in the light sensitive pool of cAMP. Thus, this receptor appears to mediate dopamine function in the cardiovascular and retinal system as well as the central nervous system.

The creation of a transfected mouse fibroblast cell line that expresses a ligand-specific receptor with the pharmacological profile of a D 2 subtype has been reported by Todd et al., (1989) Cloning of ligand-specific cell lines via gene transfer: identification of a D 2 dopamine receptor subtype Proc. Natl. Acad. Sci. USA 86:10134-10138. As one strategy in the isolation of these sequences, a rat genomic library was screened with Tm-specific probes derived from D 2 (Bunzow et al., 1988) and D 3 (Sokoloff et al., 1990) consensus sequences. Using this approach, D 1 , D 2 , and D 3 rat genomic clones, as well as a clone of an unknown receptor that had a high degree of structural identity with D 2 and D 3 receptor genes, were identified. Sequence data and pharmacological analyses demonstrated that this was the rat equivalent of the human D 4 gene of Van Tol (1991), although significant differences, as shown below, exist between the human and the rat genes. The highest levels of expression of the rat analog of the human D 4 clozapine receptor are found in the heart and the proximal aortic arch.

Isolation and Characterization of the Rat D 4 Gene

The isolation and characterization of the gene and cDNA encoding the rat D 4 dopamine receptor will be further understood by reference to the following detailed description.

Materials and Methods

Isolation of the Rat D 4 Gene

A lambda Dash rat spleen genomic library (Stratagene) was screened for D 2 -like receptor sequences with the use of radiolabeled Tm II, III, and VI/VII probes. Oligonucleotides encompassing the indicated domains were derived from consensus sequences from the rat D 2 (Bunzow et al., 1988) and D 3 genes (Sokoloff et al., 1990). Labeling of probes, hybridization, and washing were performed according to standard methodologies, for example, as described by Sambrook et al., (1989): Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, N.Y., Cold Spring Harbor Laboratory; and Feinberg and Vogelstein, (1984) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity Anal. Biochem. 137:266-267. Hybridization-positive clones were further characterized by amplification using the polymerase chain reaction (PCR) with gene-specific probes derived from coding and intron sequences of the rat D 2 gene (O'Malley et al., (1990) Organization and expression of the rat D2 A receptor gene: identification of alternative transcripts and a variant donor splice site Biochemistry 29:1367-1371), the rat D 3 gene (Sokoloff et al., 1990), and the rat D 1 gene (Monsma et al., 1990). Hybridization-positive, PCR-negative clones were plaque purified and further characterized. A 3.8-kb BamHI fragment common to the seven clones that were Tm VI/VII-positive but not identified as D 2 , D 3 , or D 1 , was subcloned and sequenced using the method of Chen and Seeburg (1985) Supercoil sequencing: a fast and simple method for sequencing plasma DNA DNA 4:165-170. DNA sequence analysis was performed with computer programs generated by Intelligenetics. The reported sequences are available from GenBank under accession number M84009.

The derived gene sequence for rat dopamine D 4 receptor is shown as Sequence ID No. 1. The deduced amino acid sequence is shown as Sequence ID No. 2.

Expression Vectors

A full-length D2 A444 cDNA clone (2.3 kb) was isolated from a rat striatal library and subcloned into the HindIII site of pcDNA/neo. A genomic D 4 fragment generated by partial NarI digestion and complete BamHI digestion was made blunt ended and ligated into the EcoRV site of pcDNA/neo. The D 4 gene in the expression vector started at nucleotide 5 and stopped 336 bp 3 of the stop codon. DNA was purified as described by Gandelman et al., (1990) Species and regional differences in the expression of cell type specific elements at the human and rat tyrosine hydroxylase gene loci J. Neurochem. 55:2149-2152, for transfection.

Cell Culture and Transfection

Mouse CCL1.3 tk fibroblasts were grown in DMEM media supplemented with 10% fetal bovine serum. Cells were plated at a density of 3 10 6 cells/10 cm dish, 12 to 24 h prior to transfection. Each plate of cells was transfected with 20 g of plasmid DNA by CaPO 4 precipitation, using the method of Chen and Okayama (1987) High efficiency transformation of mammalian cells by plasmid DNA Mol. Biol. 7:2745-2752. Four hours after transfection, cells were shocked with 20% glycerol in DMEM for 2 min, and 48 h later the cells were split and placed in fresh media supplemented with 400 g/ml of G418 (Geneticin, Gibco, active concentration). After two weeks, G418-resistant colonies were isolated with micropipette tips and screened for expression of D2 A444 or D 4 mRNA by reverse transcription/PCR analysis. Subclones expressing high levels of D2 A444 or D 4 mRNA were expanded and further characterized.

Binding Studies

Mouse fibroblasts expressing D2 A444 and D 4 were grown to 70% confluence and then harvested by scraping. After they had been washed twice in PBS, the cell pellets were resuspended in distilled water and ruptured by homogenization with a Brinkman Polytron, at setting 6 for 10 sec. Nuclei were removed by centrifugation for 5 min at 600 g. Membranes were pelleted by centrifugation for 25 min at 50,000 g. The pellets were resuspended in water and frozen at 70 C. until assayed. For receptor binding assays, samples containing 150 g of membrane protein were aliquoted into glass test tubes. 3 H -Spiperone (1 nm) and varying concentrations of competing compounds were added in a final volume of 1 ml and a final buffer of 1.5 Mm CaCl 2 , 5 mM MgCl 2 , 5 mM KCl, 120 mM NaCl, 50 mM Tris-HCl, pH 7.4 at 20 C. The tubes were incubated for 15 min at 37 C., and the assays were terminated by addition of 5 ml of ice-cold 50 Mm Tris-HCl buffer (pH 6.9), collected onto glass-fiber filters, and washed twice with the same cold buffer in a modified Brandel cell harvester. The radioactivity retained on the filters was counted in a Beckman LS 1701 scintillation counter.

Oligonucleotide primers were synthesized on an Applied Biosystems synthesizer. The Tm VI/VII primer set included orD-403, 5 -TGCTGGCTGCCCTTCTTC-3 (Sequence ID No. 5), which is identical to sequences within Tm VI in both D 2 and D 3 genes, and orD-404, 5 -GAAGCCTTGCGGAACTC-3 (Sequence ID No. 6), which is complementary to sequences from TM VII. For tissue distribution studies total RNA was reverse transcribed using orD 4 -515, 5 -CTGTCCACGCTGATGGCG-3 (Sequence ID No. 7), which is complementary to nucleotides 366 to 383 shown in Sequence ID No. 1. Second strand synthesis and further amplification utilized orD 4 -465 and orD 4 -466, 5 CAGACACCGACCAACTA-3 (Sequence ID No. 8), which is identical to nucleotides 187 to 204. Additional oligonucleotides included orD 4 -474. 5 -TGACACCCTCATGGCCAT-3 (Sequence ID No. 9), which is identical to nucleotides 309 to 326; orD 4 -465, 5 -TTGAAGATGGAGGGGGTG-3 (Sequence ID No. 10), which is complementary to nucleotides 342 to 359; orD 4 -501, 5 -GCACACCAAGCTTCACAG-3 (Sequence ID No. 11), which is identical to nucleotides 657 to 674; and orD 4 -506, 5 -TTGAAGGGCACTGT-TGACATAGC-3 (Sequence ID No. 12), which is complimentary to nucleotides 1064 to 1085. Oligonucleotides used for in situ hybridization included orD-502, 5 -ATGGTGTTGGCAGGGAAC-TCGCTC-3 (Sequence ID No. 13), which is identical with nucleotides 124 to 193, and orD-499, 5 -GAGCGAGTTCCCTGCCAACACCAT-3 (Sequence ID No. 14), which is complementary to the same nucleotides.

mRNA Analysis by PCR

Total RNA was isolated from various tissues using the method of Chomczynski and Sacchi (1987) Single-step method of RNA isolation by acid guanidium thiocyanate-phenol-chloroform extraction Anal. Biochem. 162:156-159, reverse transcribed using the method of Krug and Berger (1987) First-strand cDNA synthesis primed with oligo(dT) Methods Enzymol. 152:316-325, and further amplified as described by O'Malley et al. (1990). Amplification temperatures were: denaturation at 93 C. for 1 min, annealing at 52 C. for 1 min, and synthesis at 72 C. for 1 min for 30 cycles. PCR products were transferred to nylon after electrophoresis in 5% polyacrylamide gels. Filters were probed with an end-labeled oligonucleotide, orD 4 -474, corresponding to sequences that are internal to the amplification set. Filters were hybridized and washed according to the manufacturer's protocol (Schleicher and Schuell). The mRNA levels were normalized for equal amounts of the 18S fragment of ribosomal RNA by Northern blotting followed by hybridization with an 18S gene fragment, using the method of Chan et al., (1984) The nucleotide sequence of a rat 18S ribosomal ribonucleic acid gene, and a proposal for the secondary structure of 18S ribonucleic acid J. Biol Chem 259:224-230.

In Situ Hybridization

Sense and antisense D 4 oligonucleotide probes were end-labeled with digoxigenin-derivatized dUTP according to the manufacturer's protocols (Boehringer-Mannheim). Hybridization conditions were essentially as described by Springer et al., (1991) Non-radioactive detection of nerve growth factor receptor. (NGFR) mRNA in rat brain using in situ hybridization histochemistry J. Histochem. Cytochem. 39:231-234. The probe concentration was 35 ng/ml, and hybridization was overnight at 37 C. The final stringency wash was 0.5 SSC, 22 C. for 30 min.

Results

Low stringency screening of genomic libraries with Tm-specific probes was performed in order to isolate additional members of the dopamine D 2 receptor family. Because there are regions of very high identity between D 2 and D 3 , notably within Tm domains II and III (100% amino acid identity) and TM VI and VII (70% and 87% amino acid identity, respectively), it was reasoned that DNA fragments specific for the region encoding each transmembrane sequence might be useful in identifying other members of the D 2 -like receptor family. In addition, a comparison of the structure of the rat D 2 gene (O'Malley et al., 1990) with that of the D 3 gene (Sokoloff et al., 1990; Giros et al., (1991) Shorter variants of the D 3 dopamine receptor produced through various patterns of alternative splicing. Biochem. Biophys. Res. Commun. 176:1584-1592) indicated a high degree of conservation in the intron-exon boundaries of these genes. Therefore, Tm specific probes were constructed so as to not cross these sites, so that genomic DNA could be used as a template. Domain-specific oligonucleotides were synthesized and DNA amplification methodology used to create double-stranded DNA starting with rat genomic DNA as a template. Fragments of the appropriate size were gel purified, radiolabeled, and hybridized to a rat genomic library. The number of phage screened corresponded to 15 rat genomes of inserted DNA.

The results of screening the same set of filters successively with the Tm II, III, and VI/VII probes are shown in Table 1.

TABLE 1 Results of screening a rat genomic library with degenerate oligonucleotides encompassing transmembrane domains II, III, and VI/VII. Number of clones of each type identified by PCR* Probe D1 D2 D3 D4 TM II 13 38 24 0 TM III 7 43 20 0 TM VI/VII 0 21 25 7 *The receptor types of hybridization-positive plaques were identified with the use of gene-specific oligonucleotides in combination with PCR. The feasibility of the strategy is evident from the detection of D 1 and D 3 using the indicated probes. No unknown dopamine receptors were detected with the Tm II and III probes, suggesting either that additional receptors have less identity in these domains or that these probes are too biased towards D 2 /D 3 sequences. Probes to Tm domains I, IV, and V were not made, since there is only limited identity between D 2 and D 3 in these regions and no conservation of splice boundaries. Instead, the library was screened with the Tm VI/II probe. The majority of the 53 positive clones were D 2 and D 3 . However, seven clones were clearly different and were further characterized. DNA from the seven isolates was digested with several restriction enzymes, blotted, and probed with the TM VI/VII probe. The smallest hybridizing fragment was subcloned into Bluescript and sequenced with the Tm VI/VII PCR primers.

Translated sequences revealed two hydrophobic domains that had 62% and 64% identity to D 2 and D 3 , respectively, as well as a splice site in exactly the same position as in these genes, as described by Sokoloff et al., 1990; and O'Malley et al., (1990). Specific primers were designed from this sequence and used in unsuccessful screens of several cDNA libraries by the polymerase chain reaction (PCR), including libraries prepared from basal ganglia, hypothalamus, fetal brain, and pituitary. Subsequently, a battery of D 2 and D 3 primers derived from Tm domains I through V against the D 4 genomic clone were tested. They all seemed to hybridize to the same 3.8-kb BamHI fragment containing Tm regions VI and VII, suggesting that the new gene was very small in comparison with D 2 and D 3 . Further sequence data confirmed this premise, revealing an overall gene structure of four exons and three introns spanning approximately 3500 bp, as shown in FIG. 1 B and Sequence I.D. No. 1.

Comparison of the structural features and nucleotide sequence of the human D 4 receptor (Sequence ID No. 3, nucleotide sequence, and 4, deduced amino acid sequence) isolated from a human neuroblastoma cell line, as described by Van Tol and coworkers (1991), indicated that the new receptor is the rat analog of the D 4 receptor. The pharmacological profile of the human clone has confirmed its D 2 -like nature and suggested that this receptor has a very high affinity for clozapine (5- to 15-fold higher than the D 2 receptor; Bunzow et al., (1988); Van Tol et al., 1991).

In the coding regions, the rat gene (Sequence ID No 1) shares 73% amino acid and 77% nucleic acid sequence homology with the human D 4 gene (Sequence ID No 3). In contrast, the rat and human D 2 receptors share 95% amino acid and 90% nucleic acid identity (Mack et al., (1991) The mouse dopamine D2 A receptor gene: sequence homology with the rat and human genes and expression of alternative transcripts J. Neurochem. 57:795-801). As shown in FIG. 2 , there is between 89% and 96% identity within the transmembrane domains of these genes. Most of the differences between rat and human D 4 genes occur in the third intracytoplasmic loop where there is only 50% amino acid identity. In the human, this region encompasses an unusual splice junction within intron 3 of the D 4 gene: instead of a canonical GT/AG donor/acceptor site, a TC/CT is indicated, as reported by Van Tol et al., (1991). This unconventional splice site is not observed in the rat gene. Subsequently, Van Tol et al., (1992) have modified their interpretation of the human D4 gene structure. The human and rat genes are now predicted to have the same number of introns and exons.

The strategy depicted in FIG. 3A was used in order to rule out the presence of a small intron, less than 30 bp, with a different unusual splice site. Oligonucleotides were chosen flanking the bona fide slice site within Tm VI (o506) and the putative splice site within the third cytoplasmic loop (o501). Amplification of genomic DNA would result in a 618-bp fragment when these primers are used. The proposed model of the rat D 4 gene predicts a 426-bp band for the cDNA. Rat atrial RNA was reverse transcribed with the use of primer o506, then PCR amplification was performed with the 501/506 primer set. A single band of 426 bp was obtained, which was subcloned and sequenced. FIG. 3B demonstrates the presence of the Tm VI splice site and the absence of any additional splice sites within this sequence. Therefore, the rat D 4 gene has four exons encoding an open reading frame of 368 amino acids.

Pharmacological Confirmation that the Putative Rat D 4 Gene Codes for A D 4 Receptor

To confirm that the putative rat D 4 gene codes for a dopamine receptor analogous to the human D 4 receptor, the rat gene was inserted into the expression vector pcDNA/neo and transfected into the CCL1.3 fibroblast cell line, which was then screened for 3 H -spiperone binding. Transfected cells were enriched by expansion in medium containing G418. The rat D 2 cDNA and the human D 3 cDNA were expressed in the same cell line. The results of the 3 H -spiperone binding studies are shown in FIGS. 4A , 4 B, and 4 C.

As determined by displacement with 1 M eticlopride, specific binding of 2 nM 3 H -spiperone was about 50% of the total bound counts for all three receptors. Similar to the results reported by Van Tol et al. for the human receptors, the rat D 2 receptor has a higher affinity for eticlopride and ( )butaclamol while the rat D 4 receptor has a 2- to 3-fold higher affinity for clozapine. The human D 3 receptor also has a higher affinity for eticlopride and ( ) butaclamol and a much lower affinity for clozapine than the rat D 4 receptor. The relative rank order potency of these three compounds for the three receptors, however, demonstrates that the rat D 4 gene codes for a dopamine receptor analogous to the human D 4 gene.

Distribution of D 4 mRNA

Total RNA (250 g) from the indicated regions was isolated, reverse transcribed, and amplified using primers flanking the first intron. PCR products were separated by electrophoresis, blotted onto nylon filters, and hybridized with an oligonucleotide internal to the PCR primers. Regions tested include adrenal medulla, adrenal cortex, occipital cortex, temporal/parietal cortex, frontal cortex, olfactory bulb, basal ganglia, hippocampus, medulla, thalamus, cerebellum, and mesencephalon. One microgram of hypothalamic total RNA and 250 ng of atrial and ventricle RNA were treated as described above, except that primer or D4-465 was used. This primer generates a 171-bp product.

Various CNS and peripheral tissues were examined for the presence and relative abundance of D 4 transcripts. Total RNA was reverse transcribed with a D 4 exon 2-specific primer, and this procedure was followed by second strand synthesis and DNA amplification. The predicted D 4 PCR product of 195 bp is detected in only a few central nervous system regions such as olfactory bulb, frontal cortex, and hypothalamus. Surprisingly, the D 4 PCR product is at least 20-fold more abundant in heart than in the CNS but is not detectable in liver, adrenal cortex, adrenal medulla, or kidney. Within the heart, D 4 is more abundant in the atrial/large vessel region. In separate experiments, mouse retinas were examined for the presence of D 4 transcripts and D 4 mRNA was found to be abundantly expressed in this tissue as well (Cohen, A. I., et al., (1992) Photoreceptors of mouse retinas possess D 4 receptors coupled to adenylate cyclase Proc. Natl. Acad. Sci. USA 89, 12093-12097).

To confirm and extend these results, digoxigenin-labeled oligonucleotide probes were used for in situ hybridization histochemistry. Sense and antisense oligonucleotides were end-labeled with digoxigenin-derivitized dUTP and hybridized to 20 m frozen sections of heart and brain from 6- to 8-week-old male Sprague-Dawley rats. The hybridized oligonucleotides were visualized by alkaline phosphatase-linked anti-digoxigenin antisera and counterstained with eosin. Color development was for 16 h. Sections through the proximal aorta show intense staining of aorta. Color development was for 22 h. There was scattered hypothalamic staining in the region of the arcuate nucleus and the ventromedial nucleus of the hypothalamus, only a few positive cells in the striatum, no positive cells in the hippocampus, and the scattered presence of positive cells in the thalamus.

In the CNS, D 4 mRNA-positive cells were found primarily in hypothalamic areas surrounding the third ventricle. The hypothalamic distribution overlaps, but is not restricted to, the A11, A13, and A14 groups of hypothalamic dopaminergic cell bodies. Few positive cells were observed in the basal ganglia, hippocampus, or cortical regions. In the heart, heavily labeled cells predominated in the proximal aorta and the outflow tract of the left ventricle, with scattered positive cells throughout the central fibrous body. With more sensitive color development conditions, the predominant atrial expression of D 4 mRNA detected by PCR is evident. The distribution of staining is most consistent with the expression of D 4 mRNA in vascular smooth muscle and cardiac myocytes. D 4 mRNA was also identified in the retinal neuronal and photoreceptor layers in mouse (Cohen, et al., 1992).

The relatively high affinity of the human D 4 receptor for the neuroleptic drug clozapine, as reported by Van Tol et al., (1991), has generated interest in the possibility that this site is responsible for clozapine's novel antipsychotic effects. Clozapine also has significant tachycardia and hypotensive side effects. These have generally been ascribed to antagonist interactions at muscarinic acetylcholine receptor sites, as reported Fitton and Heel, (1990) Clozapine. A review of its pharmacological properties, and therapeutic use in schizophrenia Drugs 40:772-747. In the periphery, however, relatively selective D 2 -like agonists, such as piribedil, can cause vasodilation, hypotension, and bradycardia, as reported by McCoy et al., (1986) Selective antagonism of the hypotensive effects of dopamine agonists in spontaneously hypertensive rats Hypertension 8:298-302. These effects appear to be due at least in part to inhibition of sympathetic nerve activity, Hohli et al., (1989), and can be blocked by peripheral D 2 -like antagonists such as domperidone. Within the heart, D 2 -like receptor stimulation has positive inotropic effects, Zhao et al., (1990) Effects of dopamine D 1 and dopamine D 2 receptor agonists on coronary and peripheral hemodynamics Eur. J. Pharmacol. 190:193-202. The demonstration of high levels of expression of D 4 mRNA in the cardiovascular system indicates that some of these effects may be secondary consequences of binding to peripheral D 4 Dopamine receptors and that clozapine may be a prototypic model for a new class of receptor-selective agents for the treatment of cardiovascular disorders. Of particular interest is the recent mapping of the locus for a familial form of the long Q-T syndrome to the vicinity of H-ras-1 on chromosome 11p, as reported by Keating et al., Linkage of a cardiac-arrhythmia, the long QT syndrome, and the Harvey Ras-1 gene Science 252:704-706 (1991). This is also the location of the human D 4 receptor gene, as reported by Gelernter et al., DrD4, the D 4 dopamine receptor, maps to distal 11p Am. J. Hum. Gen. 49:340 (1991). It is possible that an abnormality of the D 4 receptor may be responsible for this cardiac conduction disorder.

In summary, it was found that the rat D 4 receptor mRNA was expressed at low levels in several central nervous system regions but at much higher levels in the heart and retina. In situ hybridization studies are consistent with a hypothalamic autoreceptor function for the D 4 receptor in the central nervous system. The major site of expression, however, was in atrial and vascular myocytes. Therefore, the D 4 receptor, unlike the other D 2 -like subtypes, may be predominantly a peripheral dopaminergic receptor. Accordingly, this receptor should be useful as a specific receptor for dopamine antagonists such as clozapine as well as dopamine agonists. By virtue of this specificity, many of these compounds can be used as regulators of blood pressure and heart rate. Depending on whether such compounds are agonists or antagonists, blood pressure may be raised or lowered and heart rate slowed or quickened. In addition, such compounds would increase or decrease, respectively, the efficiency of cardiac contractions (i.e., positive or negative inotropic effects). Similarly, such agonists and antagonists would decrease or increase light-sensitive pools of cAMP in retinal photoreceptors and effect the functioning of the eye. Effective dosages are determined based on the known dosages for these compounds for treatment of other disorders, screening for binding to cells expressing D 4 receptors, extrapolation to treatment of specific conditions, and other techniques known to those skilled in the art.

As discussed above, molecular cloning studies have defined a family of dopamine D 2 -like receptors (D 2 , D 3 , D 4 ), which are the products of separate genes. Stimulation of dopamine D 2 -like receptors in cultures of fetal cortical neurons increases the extension and branching of neurites (Todd, R. D. (1992) Biol. Psych 31, 794-807). To determine which D 2 -like receptors possess morphogenic potentials, a clonal mesencephalic cell line (MN9D) was transfected with D 2 , D 3 , or D 4 receptor subtypes, treated with the D 2 agonist quinpirole, and changes in morphology quantitated.

The results demonstrated that stimulation of D 2 receptors increased the number and branching of neurites, with little effect on neurite extension, while stimulation of D 3 and D 4 receptors increased the branching and extension of neurites. These effects on neuronal morphology could be blocked by the dopamine D 2 -like receptor antagonist eticlopride. These results suggest that all of the known D 2 -like receptors may have specific developmental roles in regulating neuronal morphogenesis of dopaminergic pathways. The types of morphological effects seen suggest that developmental abnormalities of stimulation of these receptor subtypes may result in the neuroanatomical changes found in many neurological and psychiatric disorders such as mental retardation syndromes, schizophrenia, affective disorders and autism. Regulation of receptor subtype stimulation by agonists or antagonists during pre- or postnatal life may therefore be an effective form of treatment to prevent or reverse the development of anatomical abnormalities and these diseases.

Methods

Transfection

Different dopamine receptor cDNAs or genes were transfected into the dopamine containing mesencephalic cell line, MN9D. MN9D is a cell line produced by fusion of fetal mouse mesencephalic cells with N18TG2 neuroblastoma cells, described by Choi, H., et al. (1991) Brain Res. 552, 67-76. The MN9D cell line is a stable immortalized clonal cell line established by fusion of the neuroblastoma cell N18TG2 with embryonic mouse mesencephalic dopamine producing neurons. Some of the characteristics of these cells include: the synthesis and release of dopamine; neurite formation and immunoreactivity; production of large voltage-sensitive sodium currents generated by depolarization; sensitivity to MPTP, a dopaminergic neurotoxin, and the ability to distinguish between the presence of dopaminergic target and non-target cells. Additionally, neither the MN9D nor the CCL1.3 cell lines have detectable mRNA or expressed protein for any of the D 1 -like or D 2 -like receptors.

The exon 6 containing form of the rat D2 receptor (D2 444 ) (O'Malley, et al., Organization and Expression of the rat D 2A receptor gene: identification of alternative transcripts and a variant donor splice site Biochem. 29:1367-1371 (1990)) (Sequence ID No. 15) and the human D3 receptor cDNA (Giros, et al., C.R. Acad. Sci. (Paris) III, 311, 501-508 (1990)) (Sequence ID No. 16) were inserted into the mammalian expression vector pcDNA/neo (Invitrogen). The entire rat D4 (Sequence ID No. 1) receptor gene was inserted into the same vector. All three plasmids were transfected into MN9D cells using the glycerol shock/calcium phosphate technique of Wigler, M., et al. (1979) Cell 16, 777-786; and Graham, F. and van der Eb, A (1973) Virology 52, 456-467, and permanent, clonal transfectants selected by G-418 resistance and limiting dilution. The clonal cell lines were assayed for expression of receptor mRNAs by reverse transcription of total cellular RNA with receptor specific oligonucleotides, followed by DNA amplification (O'Malley, K. L., et al, 1990) (RT/PCR) and for receptor protein by 3 H -spiperone binding (Todd, R. D., et al, 1989). Each clonal cell line expressed only the transfected dopamine receptor mRNA and the expressed receptor proteins displayed the predicted pharmacological differences for D 2 , D 3 , and D 4 receptors. The average number of expressed receptors per cell for the D 2444 , D 3 , and D 4 expressing cell lines were about 45,000, 15,000, and 3,500 respectively.

Morphology

The parental and transfected cell lines were plated at low density (8 cells/mm 2 ) onto poly(D-)lysine coated 35 mm culture dishes (Corning). The medium was Dubecco's Modified Eagle's Media (Gibco) containing 10% fetal bovine serum. 0.05% (w/v) G-418, an antibiotic which selects for the transfected cells, was added to the medium for selection. The cells were cultured in a humidified incubator under 10% CO 2 with or without 1-2 M quinpirole, a non-toxic D2-like receptor agonist.

To determine whether the stimulatory effects of quinpirole on neurite outgrowth could be blocked if mediated by dopamine D 2 -like receptors, cells were co-cultured with 2 M quinpirole and 1 M eticlopride, a D 2 -like receptor antagonist. K i s (nM) for quinpirole are 4700 82 (D2 444 ), 1567 247 (D3), 453,3 71.0 (D4); K i s(nM) for eticlopride 0.029 0.004 (D2 444 ), 0.46 0.12 (D3) 22.3 1.9 (all values are mean SEM of triplicate determinations for three to five individual assays).

Based on the K i values, the micromolar concentrations of both quinpirole and eticlopride were expected to have stimulatory or inhibitory effects at each of the D 2 -like receptors, respectively.

Cells were plated at low density, cultured overnight without treatment, then drugs or medium were added to the cultures. Quinpirole was added every 12 hours since this reagent quickly oxidizes. Eticlopride was added every 24 hours, and the control cultures received an equal amount of medium at the same time as the quinpirole additions. Living cells were photographed after 90 to 115 hours in culture using phase contrast microscopy (Nikon Diaphot) or a digital image processing system (Image-1). Morphologies of individual cells were quantitated at 1600 using a computer-interfaced drawing system with a digitizing light pad (Bioquant) as described by Todd, 1992; Sikich, L., Hickok, J. M., and Todd, R. D. Dev. Brain Res. 56, 269-274, 1990). Cells were measured consecutively over two to three dishes without knowledge of the treatment condition. Processes shorter than 5 m were not reliably remeasured and were excluded from morphometric analysis.

Results

Transfection and subsequent agonist stimulation of dopamine D2 A444 , D 3 , and D 4 receptors in MN9D cells results in distinct changes in cell morphology. These persist for at least seven days in culture and can be blocked by dopamine D2-like antagonists.

The MN9D parent cell line which does not express either D 1 - or D 2 -like receptors, and the transfected cells lines expressing D2 A444 , D 3 and D 4 receptors, elaborate neurites in culture. Unstimulated cells can be distinguished from one another less than 12 hours after plating and all the cell lines continue to develop in morphologically distinct manners for at least two weeks in culture. As shown for a single experiment in Table 2 (minus quinpirole), D2 A444 expressing cells tend to have more neurites and a larger neuritic extent. D 3 expressing cells have marked increases in neurite number, branch number and total neuritic extent, and D 4 expressing cells most closely resemble the parent MN9D cell line.

The transfected dopamine D2-like receptor expressing cell lines have distinct morphologies. Since the parent MN9D cell line synthesizes and releases dopamine (Choi, H. K. Won, L. A., Kontur, L. A., et al, (1991) Brain Res. 552, 67-76) these differences may be secondary to auto-stimulation of the expressed receptors by release of endogenous dopamine. To test whether further stimulation of receptors would result in increased morphological differences, cells were cultured in the presence of the dopamine D 2 -like agonist quinpirole. This agonist was chosen for its high affinity for all three receptors and its low toxicity to neuronal cells.

Culture with quinpirole resulted in increased morphological differences between the transfected and parent cell lines. Differences between cell lines were apparent within 24 hours of exposure to quinpirole and lasted for at least 115 hours. Table 2 shows the results of a single experiment in which MN9D parent cells and the cell lines transfected with D2 A444 , D 3 and D 4 receptors were cultured for 91 to 93 hours with and without 2 M quinpirole. Compared to MN9D cells, stimulation of the transfected cell lines with quinpirole resulted in significant increases in the number of branches and extension of neurites (Table 3). Stimulation of D2 A444 receptors resulted in a four fold increase in the frequency of branching of neurites with little effect on neurite extension (n 100 cells for each condition, significance levels given in Table 3). D 3 receptor stimulation resulted in increases in neurite branching and neurite length (n 59 cells for each condition). D 4 receptor stimulation resulted in a small increase in neurite branching and a large increase in neurite extension (n 100 cells for each condition). These effects have been observed in three independent experiments for each receptor expressing cell line, as depicted in FIGS. 5A , B, C, and D. Treatment of the MN9D parent cells with 2 M quinpirole resulted in no increases in neuritic outgrowth with the exception of the neurite number. As shown in Table 2, in this experiment there was a small but statistically significant increase in neurite number. However, in two other experiments, no significant differences in any morphological parameter were found on stimulation of MN9D cells (n 100 cells per condition for each experiment).

The effects of quinpirole stimulation varied with receptor subtype, as compared to the unstimulated state of each cell line. As shown in FIGS. 5A , 5 B and 5 C, D2 A444 stimulation resulted in significant percent increases in neurite number and branch number with only a small increase in neurite length. D 4 stimulation resulted in the largest percent increase in neurite length with no effect on neurite number. D 3 stimulation resulted in marked increases in branch number and neurite length with no effect on neurite number.

In summary, though the D 3 expressing MN9D cell line had more differentiated neurites in the unstimulated state, the D 2444 expressing cell line showed a larger increase in neurite length following quinpirole stimulation. On average, as compared to untransfected MN9D cells, the morphological effects of transfection and stimulation are that D2 A444 expressing MN9D cells have more branched neurites while D 4 expressing cells have longer less branched neurites. Stimulated, D 3 expressing MN9D cells have the most highly branched neurites and the longest total neuritic extents. The effects of quinpirole stimulation of all three receptors can be blocked by 1 M eticlopride. Similar results have been found for quinpirole stimulation of primary cultures of dopaminergic, mesencephalic neurons which express a mixture of receptor subtypes, as shown by FIG. 6 .

In conclusion, dopamine D 3 -like receptors transfected into a clonal mesencephalic cell line regulate neurite outgrowth in these cells and the D 2 receptor subtypes appear to modulate different aspects of neurite outgrowth. These results suggest a role for dopamine D 2 -receptors in mammalian neurodevelopment and provide support for the possibility that dopamine receptor subtypes differentially modulate neurite outgrowth in vivo. The observation of similar effects on neurite outgrowth in primary mesencephalic cultures supports the relevance of these effects for normal and abnormal brain development.

Assuming these responses occur in vivo, then fundamental changes in the anatomy and function of mesocortical and mesostriatal pathways could occur via abnormal receptor stimulation. These are the same brain regions where neuropathological and neuroimaging abnormalities have been reported for disorders such as schizophrenia (Benes, F. M., Davidson, J. and Bird, E. D. (1986) Arch. Gen. Psychiatr. 43, 31-35; Jeste, D. V. and Lohr, J. B. (1989) Arch. Gen. Psychiatr. 46, 1019-1024; Pfefferbaum, A. et al. (1988) Arch. Gen. Psychiatr. 45, 633-640. The results also indicate that developmental stimulation or inhibition of dopamine receptor subtypes via drugs, both therapeutically and abusively, can result in profound pre- and postnatal changes in neuronal morphology and function. Developmental regulation of receptor stimulation therefore offers a therapeutic approach to preventing or reversing neuroanatomical changes associated with a variety of neurological and psychiatric diseases.

Use of the Rat D 4 as a Screen for Cardiovascular Drugs and Other Biologically Useful Compounds

The cDNA or gene encoding the D 4 dopamine receptor can be expressed in a variety of mammalian cell lines, including the fibroblast cell line described above, or in other commercially available cell lines such as Cos cells, and used to screen for compounds which bind specifically to the D 4 receptor. This is determined by comparing binding affinities for the various D 1 , D 2 , and D 3 receptors with that of the D 4 receptor, then testing in vivo those compounds which specifically bind the receptor. It can also be expressed in bacterial cells, notably E. coli , as well as other eukaryotic expression system such as Baculovirus infection of insect cells.

Based on the discovery that the D 4 dopamine receptor is predominantly associated with cardiovascular and retinal tissues, a principle use for this screen is for compounds having an effect on the cardiovasculature and retina, either dopamine antagonists or dopamine agonists, that act as vasoregulators or have ionotropic effects and that act on retinal cyclic AMP levels. Compounds which bind either the human or the rat D 4 dopamine receptor can be screened. The typical models for physiological testing of these compounds are rats, mice and dogs. Measurements can be made in intact animals, in cardiovascular and retinal tissue explants or in isolated cells.

The gene and/or cDNA can also be used to generate probes for screening in a manner similar to those methods described above for receptors other than the known D 1 , D 2 , D 3 , and D 4 dopamine receptors. Probes are created from sequences generally fourteen to seventeen nucleotides in length, and can be labelled using available technology and reagents, including radiolabels, dyes, tomography position emission labels, magnetic resonance imaging labels, enzymes, and fluorescent labels. Probes can be used directly or indirectly via standard methodologies including polymerase chain reaction (PCR) and methodologies to generate larger fragments of the D4 receptor. Starting with either RNA (via RT/PCR) or DNA, the D4 cDNA, and parts therein, can also be used to generate RNA transcripts if cloned into appropriate expression vectors (cRNAs).

D4 DNA fragments, oligonucleotide probes or cRNAs, could all be used in commercial kits or sold separately to measure D4 transcript levels using standard techniques including PCR, in situ hybridization, and RNAse or SI protection assays.

Amino acid sequences can be deduced from fragments of D4 DNA Sequence, or the entire D4 coding sequence, generated by a variety of standard techniques for synthesis of D4 synthetic peptides, D4 fusion proteins and/or purification of D4 proteins (or parts thereof) from in vitro translated proteins derived from synthetic D4 RNA or protein purification per se. D4 proteins, peptides, fusion proteins or fragments thereof could subsequently be used for antibody production using available technology including injection into a wide variety of species including mice, rats, rabbits, guinea pigs, goats, etc. for the production of polyclonal antisera as well as injection into mice and subsequent utilization of fusion techniques for the production of monoclonal antibodies.

Oligonucleotides or larger sequences derived from the D4 mRNA or complementary sequences or antibodies directed against the D4 receptor could be labelled or derivatized to be used as imaging agents for positron emission tomography (PET) or magnetic resonance imaging (MRI) of the location of D4 receptors in vivo and in vitro.

Promoter sequences associated with the rat D4 receptor (5 flanking sequences) may be utilized to create transgenic (non-human) animals via standard methodologies, for example, by microinjection into embryos or homologous recombination in embryonic stem cells. Depending upon the reporter gene utilized (Lac Z, diptheria toxin, etc.) various animal models can be created leading to the overexpression or loss of D4 receptor activity. Additionally, promoter sequences driving foreign gene products such as the SV40 large T antigen could be used to create immortalized cell lines from D4-expressing cell types. Selected use of other foreign reporter genes (e.g. cholera toxin) can be used to make model systems whereby central nervous system or peripheral physiology can be modified. Finally, from the sequence information presented in sequences 1 and 2, vectors can be generated for subsequent homologous recombination experiments in which the D4 gene is inactivated or knocked out , allowing determination of the physiological role of the D4 gene.

Antibodies against the D4 receptor can be used for immunocytochemical localization, flow cytometry identification and isolation of D4 receptor expressing cells. Antibodies can also be used to block or modify the effects of D4 receptor agonists and antagonists both in vivo and in vitro.

The present invention is further understood by reference to the following nucleotide and amino acid sequences.

Sequence 1 is the nucleotide sequence for both the non-coding and protein coding regions of the rat D4 receptor gene loci.

Sequence 2 is the derived amino acid sequence for the rat D4 receptor protein.

Sequence 3 is the nucleotide sequence for the human D4 receptor cDNA.

Sequence 4 is the derived amino acid sequence for the human D4 receptor protein.

Sequences 5-14 are oligonucleotide primers used in the isolation of the rat D4 receptor gene.

Sequence 15 is the nucleotide sequence for the rat D2 receptor cDNA (444 amino acid form).

Sequence 16 is the nucleotide sequence for the human D3 receptor gene.

Modifications and variations of the present invention will be obvious to those skilled in the art from the foregoing detailed description. Such modifications and variations are intended to come within the scope of the following claims.