Cloning and expression of a rat D2 dopamine receptor cDNA

Dopamine receptors are classified into D1 and D2 subtypes on the basis of their pharmacological and biochemical characteristics. The D2 dopamine receptor has been implicated in the pathophysiology and treatment of movement disorders, schizophrenia and drug addiction. The D2 dopamine receptor interacts with guanine nucleotide-binding proteins to induce second messenger systems. Other members of the family of receptors that are coupled to G proteins share a significant similarity in primary amino-acid sequence and exhibit an archetypical topology predicted to consist of seven putative transmembrane domains. We have taken advantage of the expected nucleotide sequence similarities among members of this gene family to isolate genes coding for new receptors. Using the hamster beta 2-adrenergic receptor gene as a hybridization probe we have isolated related genes including a cDNA encoding the rat D2 dopamine receptor. This receptor has been characterized on the basis of three criteria: the deduced amino-acid sequence which reveals that it is a member of the family of G-protein-coupled receptors; the tissue distribution of the mRNA which parallels that of the D2 dopamine receptor; and the pharmacological profile of mouse fibroblast cells transfected with the cDNA.     

Multiple D2 dopamine receptors produced by alternative RNA splicing

Dopamine receptor belong to a large class of neurotransmitter and hormone receptors that are linked to their signal transduction pathways through guanine nucleotide binding regulatory proteins (G proteins). Pharmacological, biochemical and physiological criteria have been used to define two subcategories of dopamine receptors referred to as D1 and D2. D1 receptors activate adenylyl cyclase and are coupled with the Gs regulatory protein. By contrast, activation of D2 receptors results in various responses including inhibition of adenylyl cyclase, inhibition of phosphatidylinositol turnover, increase in K+ channel activity and inhibition of Ca2+ mobilization. The G protein(s) linking the D2 receptors to these responses have not been identified, although D2 receptors have been shown to both copurify and functionally reconstitute with both Gi and Go related proteins. The diversity of responses elicited by D2-receptor activation could reflect the existence of multiple D2 receptor subtypes, the identification of which is facilitated by the recent cloning of a complementary DNA encoding a rat D2 receptor. This receptor exhibits considerable amino-acid homology with other members of the G protein-coupled receptor superfamily. Here we report the identification and cloning of a cDNA encoding an RNA splice variant of the rat D2 receptor cDNA. This cDNA codes for a receptor isoform which is predominantly expressed in the brain and contains an additional 29 amino acids in the third cytoplasmic loop, a region believed to be involved in G protein coupling.     

Localization of D-2 dopamine receptors to intrinsic striatal neurones by quantitative autoradiography

Recent work with positron emission and single photon emission computed tomography has demonstrated the feasibility of studying striatal dopamine receptors in the living human brain. For the proper interpretation of these studies in normal and diseased states, the cellular localization of these receptors must be definitively established. It has been claimed, on the basis of receptor binding studies with tissue homogenates in rats, that 30-50% of striatal D-2 dopamine receptors are located on axons or terminals of the corticostriatal pathway. This finding has been incorporated into major reviews and classifications of dopamine receptors. The recent development of quantitative autoradiographic methods for diffusible ligands has facilitated the study of neurotransmitter receptors in cytoarchitechtonically intact tissue. Because this technique provides the necessary anatomic resolution that is lacking in homogenate binding studies, we have used it to re-examine the localization of striatal dopamine receptors. Here we present evidence that D-2 receptors are located exclusively on kainic acid-sensitive intrinsic neuronal elements in the striatum. We report that discrete cortical ablation does not alter 3H-spiperone binding to rat striatum and thus our results do not support the existence of D-2 dopamine receptors on the terminals of the corticostriatal pathway.     

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 and expression of human and rat D1 dopamine receptors

The importance of the dopaminergic system in brain function has been emphasized by its association with neurological and psychiatric disorders such as Parkinson's disease and schizophrenia. On the basis of their biochemical and pharmacological characteristics, dopamine receptors are classified into D1 and D2 subtypes. As the most abundant dopamine receptor in the central nervous system, D1 receptors seem to mediate some behavioural responses, modulate activity of D2 dopamine receptors, and regulate neuron growth and differentiation. The D dopamine receptor has been cloned by low-stringency screening. We report here the cloning of human and rat D1 dopamine receptors by applying an approach based on the polymerase chain reaction. The cloned human D1 dopamine receptor has been characterized on the basis of four criteria: the deduced amino-acid sequence, which reveals that it is a G protein-coupled receptor; the tissue distribution of its messenger RNA, which is compatible with that of the D1 dopamine receptor; its pharmacological profile when transfected into COS-7 cells; and its ability to stimulate the accumulation of cyclic AMP in human 293 cells.     

Cloning, sequence and expression of human interleukin-2 receptor

T lymphocytes, essential for the generation of a normal immune response, require the presence of the lymphokine interleukin-2 (IL-2) in order to proliferate. Cells that respond to IL-2 possess a surface receptor glycoprotein specific for this lymphokine. We have recently purified and chemically characterized the IL-2 receptor from both phytohaemagglutinin-activated human T cells and the human T-cell lymphoma HUT-102 (ref. 5). From the NH2-terminal protein sequence obtained in that study, we have now used synthetic oligonucleotides to probe a complementary DNA library, prepared from HUT-102 messenger RNA, for the presence of cDNA clones that might code for the IL-2 receptor. Two cDNA clones were isolated which had closely related DNA sequences. Interestingly, only one coded for an active receptor when transfected into COS-7 cells. This clone contained a 216-base pair (bp) insert that was not present in the other clone. The insert was flanked by an 8-bp direct repeat reminiscent of a transposable element, and appeared to code for a region of marked structural homology to the NH2-terminal region of the receptor molecule.     

Human dopamine D1 receptor encoded by an intronless gene on chromosome 5

Receptors for dopamine have been classified into two functional types, D1 and D2. They belong to the family of receptors acting through G (or guanine nucleotide-binding) proteins. D2 receptors inhibit adenylyl cyclase, but D1 receptors stimulate adenylyl cyclase and activate cyclic AMP-dependent protein kinases. Dopamine D1 and D2 receptors are targets of drug therapy in many psychomotor disorders, including Parkinson's disease and schizophrenia, and may also have a role in drug addiction and alcoholism. D1 receptors regulate neuron growth and differentiation, influence behaviour and modify dopamine D2 receptor-mediated events. We report here the cloning of the D1 receptor gene, which resides on an intronless region on the long arm of chromosome 5, near two other members of the G-linked receptor family. The expressed protein, encoded by 446 amino acids, binds drugs with affinities identical to the native human D1 receptor. The presence of a D1 receptor gene restriction fragment length polymorphism will be helpful for future disease linkage studies.     

The genomic clone G-21 which resembles a beta-adrenergic receptor sequence encodes the 5-HT1A receptor

The recent cloning of the complementary DNAs and/or genes for several receptors linked to guanine nucleotide regulatory proteins including the adrenergic receptors (alpha 1, alpha 2A, alpha 2B, beta 1, beta 2), several subtypes of the muscarinic cholinergic receptors, and the visual 'receptor' rhodopsin has revealed considerable similarity in the primary structure of these proteins. In addition, all of these proteins contain seven putative transmembrane alpha-helices. We have previously described a genomic clone, G-21, isolated by cross-hybridization at reduced stringency with a full length beta 2-adrenergic receptor probe. This clone contains an intronless gene which, because of its striking sequence resemblance to the adrenergic receptors, is presumed to encode a G-protein-coupled receptor. Previous attempts to identify this putative receptor by expression studies have failed. We now report that the protein product of the genomic clone, G21, transiently expressed in monkey kidney cells has all the typical ligand-binding characteristics of the 5-hydroxytryptamine (5-HT1A) receptor.     

Alternative splicing directs the expression of two D2 dopamine receptor isoforms

Dopamine receptors are classified into D1 and D2 subtypes on the basis of their pharmacological properties and the intracellular responses they mediate. The cerebral D2 dopamine receptor is the target of drugs used to alleviate the main symptoms of schizophrenia. Although it is considered to be a single molecular entity, there is evidence that multiple D2-receptor subtypes exist. A complementary DNA encoding a D2 receptor has recently been cloned and the deduced 415-amino-acid sequence indicates that it belongs to the large superfamily of receptors coupled to G proteins, and that its topology consists of seven transmembrane domains. In this family, the genes are frequently without introns and each is believed to encode a unique polypeptide product. Here we show that the gene for the D2 receptor produces two receptor isoforms by alternative messenger RNA splicing, providing a route to receptor diversity in this family. One isoform corresponds to the D2(415) receptor, but the second contains an additional sequence encoding a 29-amino-acid fragment, defining a novel D2(444) receptor isoform. Expression of the two isoforms is tissue-specific, and both are regulated by guanyl nucleotides. As the extra sequence is located within a putative cytoplasmic loop that binds to G proteins, the two isoforms might interact with different G proteins and thereby initiate distinct intracellular signals.     

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.     

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.     

Dopaminergic D-3 binding sites are not presynaptic autoreceptors

Postsynaptic dopamine (DA) receptors have been classified biochemically and pharmacologically into two types: D-1 receptors mediate adenylate cyclase stimulation, demonstrating micromolar affinity for DA and butyrophenone antagonists; D-2 receptors mediate adenylate cyclase inhibition, demonstrating nanomolar affinity for DA and butyrophenone antagonists. D-1 receptors are labelled by 3H-thioxanthene antagonists, while D-2 receptors are labelled by both 3H-agonists and all 3H-antagonists. A third class of dopaminergic binding site, termed D-3, represents high-affinity 3H-agonist binding sites demonstrating low, micromolar, affinity for butyrophenones. In the rat striatum, D-3 sites were decreased 50% by 6-hydroxydopamine (6-OHDA) lesions of the nigrostriatal DA pathway, suggesting that such D-3 binding labels presynaptic DA autoreceptors on nigrostriatal terminals. However, nigrostriatal denervation produces a concomitant depletion of striatal DA. Here we demonstrate that a reserpine-induced depletion of DA produces a decrease in D-3 binding comparable to that seen with nigrostriatal denervation, independent of presynaptic terminal degeneration. This loss in binding, or that caused by 6-OHDA lesions, is recovered by preincubating the striatal membranes with DA or with the supernatant from control striatal membrane preparations. We therefore suggest that the loss of D-3 binding following 6-OHDA lesions results from the depletion of endogenous DA rather than the degeneration of terminals and their putatively associated autoreceptors.     

Molecular cloning and characterization of a novel dopamine receptor (D3) as a target for neuroleptics

A dopamine receptor has been characterized which differs in its pharmacology and signalling system from the D1 or D2 receptor and represents both an autoreceptor and a postsynaptic receptor. The D3 receptor is localized to limbic areas of the brain, which are associated with cognitive, emotional and endocrine functions. It seems to mediate some of the effects of antipsychotic drugs and drugs used against Parkinson's disease, that were previously thought to interact only with D2 receptors.     

Identical V beta T-cell receptor genes used in alloreactive cytotoxic and antigen plus I-A specific helper T cells

T lymphocytes involved in the cellular immune response carry cell-surface receptors responsible for antigen and self recognition. This T-cell receptor molecule is a heterodimeric protein consisting of disulphide-linked alpha- and beta-chains with variable (V) and constant (C) regions. Several complementary DNA and genomic DNA clones have been isolated and characterized. These analyses showed that the genomic arrangement and rearrangement of T-cell receptor genes using VT, diversity (DT), joining (JT) and CT gene segments is very similar to the structure of the known immunoglobulin genes. We have isolated two cDNA clones from an allospecific cytotoxic T cell, one of which shows a productive V beta-J beta-C beta 1 rearrangement without an intervening D beta segment. This V beta gene segment is identical to the V beta gene expressed in a helper T-cell clone specific for chicken red blood cells and H-21. The other clone carries the C beta 2 gene of the T-cell receptor, but the C beta 2 sequence is preceded by a DNA sequence that does not show any similarity to V beta or J beta sequences.     

Direct protein-protein coupling enables cross-talk between dopamine D5 and gamma-aminobutyric acid A receptors

GABA(A) (gamma-aminobutyric-acid A) and dopamine D1 and D5 receptors represent two structurally and functionally divergent families of neurotransmitter receptors. The former comprises a class of multi-subunit ligand-gated channels mediating fast interneuronal synaptic transmission, whereas the latter belongs to the seven-transmembrane-domain single-polypeptide receptor superfamily that exerts its biological effects, including the modulation of GABA(A) receptor function, through the activation of second-messenger signalling cascades by G proteins. Here we show that GABA(A)-ligand-gated channels complex selectively with D5 receptors through the direct binding of the D5 carboxy-terminal domain with the second intracellular loop of the GABA(A) gamma2(short) receptor subunit. This physical association enables mutually inhibitory functional interactions between these receptor systems. The data highlight a previously unknown signal transduction mechanism whereby subtype-selective G-protein-coupled receptors dynamically regulate synaptic strength independently of classically defined second-messenger systems, and provide a heuristic framework in which to view these receptor systems in the maintenance of psychomotor disease states.     

Post-translational insertion of a fragment of the glucose transporter into microsomes requires phosphoanhydride bond cleavage

Most eukaryotic secretory and membrane proteins insert co-translationally into the membrane of the rough endoplasmic reticulum (RER), and are targeted there by one or more NH2-terminal or internal signal sequences. However, little is known about the actual translocation and membrane integration processes. In particular, any energy requirements for targeting and integration have remained obscure because of the inability to uncouple the processes from concomitant protein synthesis. We recently showed that the human glucose transporter (GT), an integral membrane glycoprotein, can insert post-translationally into dog pancreatic microsomes with low but demonstrable efficiency in vitro, and that a fragment corresponding to the NH2-terminal 340 amino acids and 8 of the 12 membrane-spanning alpha-helixes of GT (GT-N) can insert with significantly greater efficiency. We report here that post-translational insertion of GT-N into pancreatic microsomes requires energy in the form of a phosphodiester bond, and suggest that co-translational insertion of proteins into the RER may also require energy independent of that used for polypeptide synthesis.     

Control of visual cortical signals by prefrontal dopamine

The prefrontal cortex is thought to modulate sensory signals in posterior cortices during top-down attention, but little is known about the underlying neural circuitry. Experimental and clinical evidence indicate that prefrontal dopamine has an important role in cognitive functions, acting predominantly through D1 receptors. Here we show that dopamine D1 receptors mediate prefrontal control of signals in the visual cortex of macaques (Macaca mulatta). We pharmacologically altered D1-receptor-mediated activity in the frontal eye field of the prefrontal cortex and measured the effect on the responses of neurons in area V4 of the visual cortex. This manipulation was sufficient to enhance the magnitude, the orientation selectivity and the reliability of V4 visual responses to an extent comparable with the known effects of top-down attention. The enhancement of V4 signals was restricted to neurons with response fields overlapping the part of visual space affected by the D1 receptor manipulation. Altering either D1- or D2-receptor-mediated frontal eye field activity increased saccadic target selection but the D2 receptor manipulation did not enhance V4 signals. Our results identify a role for D1 receptors in mediating the control of visual cortical signals by the prefrontal cortex and suggest how processing in sensory areas could be altered in mental disorders involving prefrontal dopamine.     

Molecular cloning and expression of the gene for a human D1 dopamine receptor

The diverse physiological actions of dopamine are mediated by its interaction with two basic types of G protein-coupled receptor, D1 and D2, which stimulate and inhibit, respectively, the enzyme adenylyl cyclase. Alterations in the number or activity of these receptors may be a contributory factor in diseases such as Parkinson's disease and schizophrenia. Here we describe the isolation and characterization of the gene encoding a human D1 dopamine receptor. The coding region of this gene is intronless, unlike the gene encoding the D2 dopamine receptor. The D1 receptor gene encodes a protein of 446 amino acids having a predicted relative molecular mass of 49,300 and a transmembrane topology similar to that of other G protein-coupled receptors. Transient or stable expression of the cloned gene in host cells established specific ligand binding and functional activity characteristic of a D1 dopamine receptor coupled to stimulation of adenylyl cyclase. Northern blot analysis and in situ hybridization revealed that the messenger RNA for this receptor is most abundant in caudate, nucleus accumbens and olfactory tubercle, with little or no mRNA detectable in substantia nigra, liver, kidney, or heart. Several observations from this work in conjunction with results from other studies are consistent with the idea that other D1 dopamine receptor subtypes may exist.     

Dopamine activation of the arachidonic acid cascade as a basis for D1/D2 receptor synergism

Understanding the actions of the neurotransmitter dopamine in the brain is important in view of its roles in neuropsychiatric illnesses. Dopamine D1 receptors, which stimulate both adenylyl cyclase and phospholipase C, and D2 receptors, which inhibit them, can nevertheless act synergistically to produce many electrophysiological and behavioral responses. Because this functional synergism can occur at the level of single neurons, another, as yet unidentified, signalling pathway activated by dopamine has been hypothesized. We report here that in Chinese hamster ovary (CHO) cells transfected with the D2 receptor complementary DNA, D2 agonists potently enhanced arachidonic acid release, provided that such release has been initiated by stimulating constitutive purinergic receptors or by increasing intracellular Ca2+. In CHO cells expressed D1 receptors, D1 agonists exert no such effect. When D1 and D2 receptors are coexpressed, however, activation of both subtypes results in a marked synergistic potentiation of arachidonic acid release. The numerous actions of arachidonic acid and its metabolites in neuronal signal transduction suggest that facilitation of its release may be implicated in dopaminergic responses, such as feedback inhibition mediated by D2 autoreceptors, and may constitute a molecular basis for D1/D2 receptor synergism.