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.     

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.     

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.     

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.     

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.     

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.     

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.     

Characterization of the human cysteinyl leukotriene CysLT1 receptor.

The cysteinyl leukotrienes-leukotriene C4(LTC4), leukotriene D4(LTD4) and leukotriene E4(LTE4)-are important mediators of human bronchial asthma. Pharmacological studies have determined that cysteinyl leukotrienes activate at least two receptors, designated CysLT1 and CysLT2. The CysLT1-selective antagonists, such as montelukast (Singulair), zafirlukast (Accolate) and pranlukast (Onon), are important in the treatment of asthma. Previous biochemical characterization of CysLT1 antagonists and the CysLT1 receptor has been in membrane preparations from tissues enriched for this receptor. Here we report the molecular and pharmacological characterization of the cloned human CysLT1 receptor. We describe the functional activation (calcium mobilization) of this receptor by LTD4 and LTC4, and competition for radiolabelled LTD4 binding to this receptor by the cysteinyl leukotrienes and three structurally distinct classes of CysLT1-receptor antagonists. We detected CysLT1-receptor messenger RNA in spleen, peripheral blood leukocytes and lung. In normal human lung, expression of the CysLT1-receptor mRNA was confined to smooth muscle cells and tissue macrophages. Finally, we mapped the human CysLT1-receptor gene to the X chromosome.     

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.     

Auto-inhibition of brain histamine release mediated by a novel class (H3) of histamine receptor

Although histaminergic neurones have not yet been histochemically visualized, there is little doubt that histamine (HA) has a neurotransmitter role in the invertebrate and mammalian central nervous system. For example, a combination of biochemical, electrophysiological and lesion studies in rats have shown that histamine is synthesized in and released from a discrete set of neurones ascending through the lateral hypothalamic area and widely projecting in the telencephalon. Histamine acts on target cells in mammalian brain via stimulation of two classes of receptor (H1 and H2) previously characterized in peripheral organs and probably uses Ca2+ and cyclic AMP, respectively, as second messengers. It is well established that several neurotransmitters affect neuronal activity in the central nervous system through stimulation not only of postsynaptic receptors, but also of receptors located presynaptically which often display distinct pharmacological specificity and by which they may control their own release. Such 'autoreceptors' have been demonstrated (or postulated) in the case of noradrenaline, dopamine, serotonin, acetylcholine and gamma-aminobutyric acid (GABA) neurones but have never been demonstrated for histamine. We show here that histamine inhibits its own release from depolarized slices of rat cerebral cortex, an action apparently mediated by a class of receptor (H3) pharmacologically distinct from those previously characterized, that is, the H1 and H2 receptors.     

Cloning and characterization of a vasopressin V2 receptor and possible link to nephrogenic diabetes insipidus.

The antidiuretic effect of arginine vasopressin (AVP) is mediated by renal-type (V2) receptors linked to adenylyl cyclase. We report here the cloning of the rat kidney V2 AVP receptor complementary DNA that encodes a 370-amino-acid protein with a transmembrane topography characteristic of G protein-coupled receptors, and with similarity to the V1a (hepatic) AVP receptor in its seven membrane-spanning domains. Expression of the cloned cDNA in mammalian cells showed specific ligand binding and activity characteristic of the native V2 AVP receptor. The receptor messenger RNA is detected only in the kidney. The human V2 receptor gene has been localized to the long arm of the X chromosome close to the locus for nephrogenic diabetes insipidus, an X-linked recessive disorder characterized by renal resistance to the antidiuretic action of AVP.     

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.     

Molecular cloning and characterization of the rat NMDA receptor.

A complementary DNA encoding the rat NMDA receptor has been cloned and characterized. The single protein encoded by the cDNA forms a receptor-channel complex that has electrophysiological and pharmacological properties characteristic of the NMDA receptor. This protein has a significant sequence similarity to the AMPA/kainate receptors and contains four putative transmembrane segments following a large extracellular domain. The NMDA receptor messenger RNA is expressed in neuronal cells throughout the brain regions, particularly in the hippocampus, cerebral cortex and cerebellum.     

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.     

A single amino-acid difference confers major pharmacological variation between human and rodent 5-HT1B receptors.

Neuropsychiatric disorders such as anxiety, depression, migraine, vasospasm and epilepsy may involve different subtypes of the 5-hydroxytryptamine (5-HT) receptor. The 1B subtype, which has a unique pharmacology, was first identified in rodent brain. But a similar receptor could not be detected in human brain, suggesting the absence in man of a receptor with equivalent function. Recently a human receptor gene was isolated (designated 5-HT1B receptor, 5-HT1D beta receptor, or S12 receptor) which shares 93% identity of the deduced protein sequence with rodent 5-HT1B receptors. Although this receptor is identical to rodent 5-HT1B receptors in binding to 5-HT, it differs profoundly in binding to many drugs. Here we show that replacement of a single amino acid in the human receptor (threonine at residue 355) with a corresponding asparagine found in rodent 5-HT1B receptors renders the pharmacology of the receptors essentially identical. This demonstrates that the human gene does indeed encode a 1B receptor, which is likely to have the same biological functions as the rodent 5-HT1B receptor. In addition, these findings show that minute sequence differences between homologues of the same receptor from different species can cause large pharmacological variation. Thus, drug-receptor interactions should not be extrapolated from animal to human species without verification.     

Structure of the human M2 muscarinic acetylcholine receptor bound to an antagonist

The parasympathetic branch of the autonomic nervous system regulates the activity of multiple organ systems. Muscarinic receptors are G-protein-coupled receptors that mediate the response to acetylcholine released from parasympathetic nerves. Their role in the unconscious regulation of organ and central nervous system function makes them potential therapeutic targets for a broad spectrum of diseases. The M2 muscarinic acetylcholine receptor (M2 receptor) is essential for the physiological control of cardiovascular function through activation of G-protein-coupled inwardly rectifying potassium channels, and is of particular interest because of its extensive pharmacological characterization with both orthosteric and allosteric ligands. Here we report the structure of the antagonist-bound human M2 receptor, the first human acetylcholine receptor to be characterized structurally, to our knowledge. The antagonist 3-quinuclidinyl-benzilate binds in the middle of a long aqueous channel extending approximately two-thirds through the membrane. The orthosteric binding pocket is formed by amino acids that are identical in all five muscarinic receptor subtypes, and shares structural homology with other functionally unrelated acetylcholine binding proteins from different species. A layer of tyrosine residues forms an aromatic cap restricting dissociation of the bound ligand. A binding site for allosteric ligands has been mapped to residues at the entrance to the binding pocket near this aromatic cap. The structure of the M2 receptor provides insights into the challenges of developing subtype-selective ligands for muscarinic receptors and their propensity for allosteric regulation.     

Reconstitution of functional receptor for human interleukin-2 in mouse cells

Interleukin-2 (IL-2) has a key role in the antigen-specific clonal growth of T lymphocytes, by virtue of its interaction with a specific cell-surface receptor (IL-2R). The growth signal seems to be delivered by IL-2 bound to the high-affinity, but not the low-affinity, receptor. Genes encoding IL-2 and its receptor (that is, Tac-antigen) have been cloned and analysed in detail. We have now achieved cell-type-specific reconstitution of the high-affinity human IL-2R by expressing the complementary DNA cloned from normal lymphocytes. A mouse T-lymphocytic line, EL-4, expressed human IL-2R with high (dissociation constant (Kd) = 160-220 pM) and low (Kd = 2.1-2.2 nM) affinity for recombinant human IL-2, while mouse L929 cells expressed only a single class of the IL-2R with lower affinity (Kd = 34.5 nM) for the ligand. We also show that the human IL-2R expressed in EL-4 cells responds to IL-2 and mediates reversed signal transduction: growth of the EL-4 cells harbouring the IL-2R is inhibited specifically by human recombinant IL-2. The approach described here may provide a general experimental framework for elucidating the molecular basis of signal transduction mediated by specific receptor-ligand interaction.     

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.