ABSTRACT
Acylated and aroylated hydrazinoclozapines are highly potent dopamine D(1) antagonists that show remarkable selectivity over other dopamine receptors. The most potent compound in this series is the 2,6-dimethoxybenzhydrazide 33 with a D(1)K(i) of 1.6 nM and 212-fold selectivity over D(2) receptor.
Subject(s)
Clozapine/chemistry , Clozapine/pharmacology , Dopamine Antagonists/chemical synthesis , Dopamine Antagonists/pharmacology , Hydrazines/chemistry , Receptors, Dopamine D1/antagonists & inhibitors , Clozapine/chemical synthesis , Clozapine/classification , Dopamine Antagonists/chemistry , Dopamine Antagonists/classification , Dopamine D2 Receptor Antagonists , Molecular Structure , Receptors, Dopamine D1/metabolism , Receptors, Dopamine D2/metabolism , Structure-Activity RelationshipABSTRACT
Neurokinin receptors in the central nervous system are involved in the neural circuitry of anxiety, depression and emesis. This has led to the development of nonpeptidic NK1 receptor antagonists as therapeutic agents. Clinical trials have shown that NK1 receptor antagonists have efficacy in chemotherapy-induced emesis and depression. Sequence polymorphisms can potentially influence the efficacy of drugs in patient populations and are an important consideration in the drug development process. To identify DNA sequence variants in the NK1 receptor, comparative DNA sequencing was performed on a population of 93 individuals. In total, 19 single-nucleotide polymorphisms (SNPs) were identified with one SNP (g.78351T>C) resulting in a tyrosine to histidine substitution at residue 192 (Y192H). The Y192H variant was expressed using site-directed mutagenesis and was characterized with respect to affinity, receptor kinetics, functional calcium response and receptor internalization. In all cases the Y192H variant was found to display properties similar to those of the wild-type receptor.
Subject(s)
Amino Acid Substitution/genetics , Genetic Variation/genetics , Polymorphism, Single Nucleotide/genetics , Receptors, Neurokinin-1/genetics , Substance P/analogs & derivatives , Alleles , Animals , COS Cells , Chlorocebus aethiops , Dose-Response Relationship, Drug , Gene Frequency/genetics , Genetic Variation/drug effects , Histidine/genetics , Humans , Neurokinin-1 Receptor Antagonists , Peptide Fragments/pharmacology , Polymorphism, Single Nucleotide/drug effects , Protein Binding/drug effects , Protein Binding/genetics , Receptors, Neurokinin-1/agonists , Substance P/pharmacology , Tyrosine/geneticsABSTRACT
Hemokinin-1 (HK-1) is a recently described mouse tachykinin peptide whose biological functions are not fully understood. To date, a unique receptor for HK-1 has not been identified. Recent studies suggest HK-1 may have a role in immunological functions, but there has been little characterization of HK-1's effects in the central nervous system (CNS). In the present studies, we confirm that HK-1 is an endogenous agonist at all of the known tachykinin receptors, and is selective for the NK1 receptor over the NK2 and NK3 subtypes. CHO cells transfected with the human NK1 receptor released intracellular calcium in response to HK-1. In addition, HK-1 competed with substance P (SP) for binding to mouse NK1 and human NK1 receptors. In vivo central administration of HK-1 to gerbils and mice induced foot-tapping and scratching behaviors, respectively, similar to those observed following central administration of SP or the NK1 receptor agonist, GR-73632. Furthermore, these behavioral effects were blocked by the selective NK1 receptor antagonist, MK-869. Finally, a comprehensive expression analysis of HK-1 demonstrated that HK-1 mRNA is much more broadly expressed than previously reported with expression observed in many brain regions. Together these data demonstrate that HK-1 is a functional agonist at NK1 receptors and suggest that HK-1 may function both centrally and peripherally.
Subject(s)
Behavior, Animal/drug effects , Protein Precursors/administration & dosage , Receptors, Neurokinin-1/agonists , Substance P/pharmacology , Tachykinins/administration & dosage , Animals , Behavior, Animal/physiology , CHO Cells , Cricetinae , Dose-Response Relationship, Drug , Female , Gerbillinae , Humans , Male , Mice , Neurokinin-1 Receptor Antagonists , Protein Precursors/biosynthesis , Receptors, Neurokinin-1/metabolism , Substance P/biosynthesis , Tachykinins/biosynthesisABSTRACT
The potential toxicological liabilities of the M(2) muscarinic antagonist 1 were addressed by replacing the methylenedioxyphenyl moiety with a p-methoxyphenyl group, resulting in M(2) selective compounds such as 3. Several halogenated naphthamide derivatives of 3 were studied in order to improve the pharmacokinetic profile via blockage of oxidative metabolism. Compound 4 demonstrated excellent M(2) affinity and selectivity, human microsomal stability, and oral bioavailability in rodents and primates.
Subject(s)
Benzylidene Compounds/chemistry , Dioxoles/chemistry , Dioxoles/pharmacology , Muscarinic Antagonists/chemistry , Muscarinic Antagonists/pharmacology , Receptors, Muscarinic/drug effects , Sulfones/chemistry , Sulfones/pharmacology , Acetylcholine/analysis , Acetylcholine/metabolism , Administration, Oral , Animals , Area Under Curve , Benzylidene Compounds/metabolism , Cytochrome P-450 Enzyme System/metabolism , Drug Design , Drug Evaluation, Preclinical , Drug Stability , Humans , Macaca fascicularis , Microdialysis , Microsomes, Liver/drug effects , Microsomes, Liver/metabolism , Muscarinic Antagonists/blood , Rats , Receptor, Muscarinic M2 , Structure-Activity RelationshipABSTRACT
Current treatment of Alzheimer's Disease (AD) requires acetylcholinesterase inhibition to increase acetylcholine (ACh) concentrations in the synaptic cleft. Another mechanism by which ACh levels can be increased is blockade of presynaptic M2 muscarinic autoreceptors that regulate ACh release. An antagonist designed for this purpose must be highly selective for M2 receptors to avoid blocking postsynaptic M1 receptors, which mediate the cognitive effects of ACh. Structure-activity studies of substituted methylpiperadines led to the synthesis of 4-[4-[1(S)-[4-[(1,3-benzodioxol-5-yl)sulfonyl]phenyl]ethyl]-3(R)-methyl-1-piperazinyl]-4-methyl-1-(propylsulfonyl)piperidine. This compound, SCH 72788, binds to cloned human M2 receptors expressed in CHO cells with an affinity of 0.5 nM, and its affinity at M1 receptors is 84-fold lower. SCH 72788 is a functional M2 antagonist that competitively inhibits the ability of the agonist oxotremorine-M to inhibit adenylyl cyclase activity. In an in vivo microdialysis paradigm, SCH 72788 increases ACh release from the striatum of conscious rats. The compound is also active in a rodent model of cognition, the young rat passive avoidance response paradigm. The effects of SCH 72788 suggest that M2 receptor antagonists may be useful for treating the cognitive decline observed in AD and other dementias.
Subject(s)
Acetylcholine/metabolism , Muscarinic Antagonists/pharmacology , Piperazines/pharmacology , Piperidines/pharmacology , Receptors, Muscarinic/metabolism , Synapses/drug effects , Adenylyl Cyclases/metabolism , Alzheimer Disease/drug therapy , Animals , CHO Cells , Cricetinae , Dose-Response Relationship, Drug , Humans , Kinetics , Learning/drug effects , Memory/drug effects , Molecular Structure , Muscarinic Agonists/pharmacology , Muscarinic Antagonists/chemical synthesis , Muscarinic Antagonists/metabolism , Muscarinic Antagonists/therapeutic use , Oxotremorine/pharmacology , Piperazines/chemical synthesis , Piperazines/metabolism , Piperazines/therapeutic use , Piperidines/chemical synthesis , Piperidines/metabolism , Piperidines/therapeutic use , Radioligand Assay , Rats , Receptor, Muscarinic M2 , Signal Transduction/physiology , Synapses/metabolismABSTRACT
ADP plays a critical role in modulating thrombosis and hemostasis. ADP initiates platelet aggregation by simultaneous activation of two G protein-coupled receptors, P2Y1 and P2Y12. Activation of P2Y1 activates phospholipase C and triggers shape change, while P2Y12 couples to Gi to reduce adenylyl cyclase activity. P2Y12 has been shown to be the target of the thienopyridine drugs, ticlopidine and clopidogrel. Recently, we cloned a human orphan receptor, SP1999, highly expressed in brain and platelets, which responded to ADP and had a pharmacological profile similar to that of P2Y12. To determine whether SP1999 is P2Y12, we generated SP1999-null mice. These mice appear normal, but they exhibit highly prolonged bleeding times, and their platelets aggregate poorly in responses to ADP and display a reduced sensitivity to thrombin and collagen. These platelets retain normal shape change and calcium flux in response to ADP but fail to inhibit adenylyl cyclase. In addition, oral clopidogrel does not inhibit aggregation responses to ADP in these mice. These results demonstrate that SP1999 is indeed the elusive receptor, P2Y12. Identification of the target receptor of the thienopyridine drugs affords us a better understanding of platelet function and provides tools that may lead to the discovery of more effective antithrombotic therapies.
Subject(s)
Blood Platelets/drug effects , Fibrinolytic Agents/pharmacology , Membrane Proteins , Purinergic P2 Receptor Antagonists , Ticlopidine/pharmacology , Adenosine Diphosphate/pharmacology , Adenylyl Cyclases/metabolism , Animals , Bleeding Time , Blood Coagulation , Blood Platelets/metabolism , Cells, Cultured , Clopidogrel , Gene Targeting , Kinetics , Mice , Mice, Knockout , Platelet Aggregation/drug effects , Receptors, Purinergic P2/genetics , Receptors, Purinergic P2Y12 , Ticlopidine/analogs & derivativesABSTRACT
Novel, selective M2 muscarinic antagonists, which replace the metabolically labile styrenyl moiety of the prototypical M2 antagonist 1 with an ether linkage, were synthesized. A detailed SAR study in this class of compounds has yielded highly active compounds that showed M2 Ki values of < 1.0 nM and >100-fold selectivity against M1, M3, and M5 receptors.
Subject(s)
Acetylcholine/agonists , Ether/analogs & derivatives , Ether/pharmacology , Muscarinic Antagonists/pharmacology , Receptors, Muscarinic/drug effects , Alkenes/chemical synthesis , Drug Design , Humans , Muscarinic Antagonists/chemical synthesis , Protein Binding , Receptor, Muscarinic M1 , Receptor, Muscarinic M2 , Receptor, Muscarinic M3 , Receptor, Muscarinic M5 , Structure-Activity RelationshipABSTRACT
Identification of a number of highly potent M2 receptor antagonists with >100-fold selectivity against the M1 and M3 receptor subtypes is described. In the rat microdialysis assay, this series of compounds showed pronounced enhancement of brain acetylcholine release after oral administration.
Subject(s)
Muscarinic Antagonists/chemical synthesis , Piperidines/chemical synthesis , Receptors, Muscarinic/physiology , Acetylcholine/metabolism , Administration, Oral , Animals , Brain/drug effects , Brain/metabolism , Drug Design , Microdialysis , Muscarinic Antagonists/chemistry , Muscarinic Antagonists/pharmacology , Piperidines/chemistry , Piperidines/pharmacology , Rats , Receptor, Muscarinic M1 , Receptor, Muscarinic M2 , Receptors, Muscarinic/drug effects , Structure-Activity RelationshipABSTRACT
Structure activity studies on [4-(phenylsulfonyl)phenyl]methylpiperazine led to the discovery of 4-cyclohexyl-alpha-[4-[[4-methoxyphenyl(S)-sufinyl]phenyl]-1-pi perazineacetonitrile, 1, an M2 selective muscarinic antagonist. Affinity at the cloned human M2 receptor was 2.7 nM; the M1/M2 selectivity is 40-fold.
Subject(s)
Benzene Derivatives/chemical synthesis , Muscarinic Antagonists/chemical synthesis , Receptors, Muscarinic/drug effects , Acetylcholine/metabolism , Administration, Oral , Alkaloids/chemistry , Alkaloids/pharmacology , Animals , Benzene Derivatives/chemistry , Benzene Derivatives/pharmacology , Corpus Striatum/drug effects , Corpus Striatum/metabolism , Dibenzazepines/chemistry , Dibenzazepines/pharmacology , Drug Design , Furans , Humans , Muscarinic Antagonists/chemistry , Muscarinic Antagonists/pharmacology , Naphthalenes , Piperidines , Pyridines/chemistry , Pyridines/pharmacology , Rats , Receptor, Muscarinic M2 , Receptors, Muscarinic/physiology , Recombinant Proteins/antagonists & inhibitors , Structure-Activity RelationshipABSTRACT
Benzylidene ketal derivatives were investigated as selective M2 receptor antagonists for the treatment of Alzheimer's disease. Compound 10 was discovered to have subnanomolar M2 receptor affinity and 100-fold selectivity against other muscarinic receptors. Also, 10 demonstrated in vivo efficacy in rodent models of muscarinic activity and cognition.
Subject(s)
Benzylidene Compounds/metabolism , Benzylidene Compounds/pharmacokinetics , Receptors, Muscarinic/metabolism , Acetylcholine/metabolism , Alzheimer Disease/drug therapy , Animals , Benzylidene Compounds/chemical synthesis , Brain/metabolism , Cholinergic Agonists/chemical synthesis , Cholinergic Agonists/metabolism , Cholinergic Agonists/pharmacokinetics , Cognition/drug effects , Drug Stability , Humans , Memory/drug effects , Muscarinic Antagonists/chemical synthesis , Muscarinic Antagonists/metabolism , Muscarinic Antagonists/pharmacokinetics , Protein Binding , Rats , Receptor, Muscarinic M2 , Structure-Activity RelationshipABSTRACT
As a decrease in cholinergic neurons has been observed in Alzheimer's Disease (AD), therapeutic approaches to AD include inhibition of acetylcholinesterase to increase acetylcholine levels. Evidence suggests that acetylcholine release in the CNS is modulated by negative feedback via presynaptic M2 receptors, blockade of which should provide another means of increasing acetylcholine release. Structure-activity studies of [4-(phenylsulfonyl)phenyl]methylpiperazines led to the synthesis of 4-cyclohexyl-alpha-[4-[[4-methoxyphenyl]sulfinyl]-phenyl]-1-piperazin eacetonitrile. This compound, SCH 57790, binds to cloned human M2 receptors expressed in CHO cells with an affinity of 2.78 nM; the affinity at M1 receptors is 40-fold lower. SCH 57790 is an antagonist at M2 receptors expressed in CHO cells, as the compound blocks the inhibition of adenylyl cyclase activity mediated by the muscarinic agonist oxotremorine. This compound should be useful in assessing the potential of M2 receptor blockade for enhancement of cognition.
Subject(s)
Muscarinic Antagonists/pharmacology , Piperazines/pharmacology , Receptors, Muscarinic/physiology , Acetylcholine/metabolism , Adenylyl Cyclases/metabolism , Alzheimer Disease/drug therapy , Animals , Binding Sites , CHO Cells , Colforsin/antagonists & inhibitors , Colforsin/pharmacology , Cricetinae , Cyclic AMP/metabolism , Humans , Muscarinic Agonists/pharmacology , Muscarinic Antagonists/chemistry , Muscarinic Antagonists/metabolism , Muscarinic Antagonists/therapeutic use , Oxotremorine/pharmacology , Piperazines/chemistry , Piperazines/metabolism , Quinuclidinyl Benzilate/metabolism , Receptor, Muscarinic M2 , Receptors, Muscarinic/metabolism , TransfectionABSTRACT
Dopamine receptors belong to a large super-gene family of receptors which are linked to their signal transduction pathways through heterotrimeric G proteins. A variety of signalling events are known to be regulated by dopamine receptors including adenylate cyclase and phospholipase activities and various ion channels. Prior to the advent of molecular cloning technology, dopamine receptors were believed to belong to two subtypes, D1 and D2. This distinction was based on both pharmacological and functional criteria. We now know that at least five different dopamine receptors exist although they can still be described as to belonging within "D1" and "D2" subfamilies. The D1 subfamily consists of two receptors-the D1 and D5, whereas the D2, D3 and D4 receptors comprise the D2 subfamily. The cloning and molecular characteristics of these five receptors are described in this review.
Subject(s)
Dopamine Agonists/pharmacology , Dopamine Antagonists/pharmacology , Gene Expression Regulation/genetics , Receptors, Dopamine/genetics , Adenylyl Cyclases/metabolism , Amino Acid Sequence , Animals , Cloning, Molecular , Humans , Ion Channels/metabolism , Molecular Sequence Data , Phospholipases/metabolism , Receptors, Dopamine/biosynthesis , Receptors, Dopamine/drug effects , Receptors, Dopamine D1/drug effects , Receptors, Dopamine D1/genetics , Receptors, Dopamine D1/metabolism , Receptors, Dopamine D2/drug effects , Receptors, Dopamine D2/genetics , Receptors, Dopamine D2/metabolism , Receptors, Dopamine D3 , Receptors, Dopamine D4 , Receptors, Dopamine D5 , Signal Transduction/geneticsABSTRACT
We have sought to determine which area of the D2 dopamine receptor's third intracellular loop contributes to G-protein coupling by constructing reciprocal chimeric D2/D3 receptors with fusion points near the center of the third intracellular loop. Both receptor chimeras were expressed equally well in Chinese Hamster Ovary (CHO) cells and exhibited ligand binding properties similar to those of the wild type receptors. Surprisingly, both of the D2/D3 receptor chimeras were able to effectively inhibit adenylyl cyclase activity to almost the same extent as that seen with the D2 receptor whereas the D3 receptor was without effect. These results suggest that the D2 receptor possesses two redundant and independent domains for G-protein coupling and inhibition of adenylyl cyclase activity.
Subject(s)
Adenylyl Cyclases/metabolism , Receptors, Dopamine D2/metabolism , Recombinant Fusion Proteins/metabolism , Amino Acid Sequence , Animals , Benzopyrans/metabolism , CHO Cells , Colforsin/pharmacology , Cricetinae , Cyclic AMP/metabolism , Dopamine Agonists/metabolism , Molecular Sequence Data , Oxazines/metabolism , Protein Binding , Rats , Receptors, Dopamine D3 , Tetrahydronaphthalenes/metabolismABSTRACT
In rodents, D1 dopamine receptors are expressed in the suprachiasmatic nucleus and are believed to play important roles in regulating circadian rhythms. It is not currently known if the primate circadian system can be influenced by dopaminergic agents, which have broad clinical use. To determine if dopamine receptors can potentially influence primate circadian function, we examined the expression of D1 dopamine receptors in the anterior hypothalamus of ring-tailed macaques (Macaca nemestrema), baboons (Papto sp.), and humans. Because D5 dopamine receptors also stimulate adenylyl cyclase activity, D5 dopamine receptor expression was studied as well. We used [125I]SCH 23982, which binds to D1 and D5 dopamine receptors, and labeling of the suprachiasmatic (SCN), supraoptic (SON), and paraventricular (PVN) nuclei was detectable in each species. In situ hybridization studies revealed differential expression of D1 and D5 dopamine receptor mRNA in the hypothalamus. D1 dopamine receptor mRNA was expressed in the SCN, SON, and PVN. By contrast, D5 dopamine receptor mRNA was expressed only in the SON and PVN of baboons and humans. Injection of the D1/D5 dopamine receptor agonist SKF 38393 at night increased the uptake of 2-deoxy-D-[14C]glucose in the SCN, SON, and PVN of newborn baboons. By contrast, c-fos mRNA expression was induced in the SON and PVN, but not in the SCN. These data show that D1 and D5 dopamine receptors are present in the hypothalamus of primates and show that activation of these receptors acutely influences SCN, SON, and PVN activity.
Subject(s)
Paraventricular Hypothalamic Nucleus/metabolism , Receptors, Dopamine D1/metabolism , Suprachiasmatic Nucleus/metabolism , Supraoptic Nucleus/metabolism , Animals , In Situ Hybridization , Macaca , Papio , Receptors, Dopamine D5ABSTRACT
While most effects of dopamine in the brain are mediated by the D1 and D2 receptor subtypes, other members of this G protein-coupled receptor family have potentially important functions. D3 receptors belong to the D2-like subclass of dopamine receptors, activation of which inhibits adenylyl cyclase. Using targeted mutagenesis in mouse embryonic stem cells, we have generated mice lacking functional D3 receptors. A premature chain-termination mutation was introduced in the D3 receptor gene after residue Arg-148 in the second intracellular loop of the predicted protein sequence. Binding of the dopamine antagonist [125I]iodosulpride to D3 receptors was absent in mice homozygous for the mutation and greatly reduced in heterozygous mice. Behavioral analysis of mutant mice showed that this mutation is associated with hyperactivity in an exploratory test. Homozygous mice lacking D3 receptors display increased locomotor activity and rearing behavior. Mice heterozygous for the D3 receptor mutation show similar, albeit less pronounced, behavioral alterations. Our findings indicate that D3 receptors play an inhibitory role in the control of certain behaviors.
Subject(s)
Receptors, Dopamine D2/metabolism , Amino Acid Sequence , Animals , Base Sequence , Behavior, Animal/physiology , Brain/physiology , DNA Primers/chemistry , Mice , Mice, Knockout , Molecular Sequence Data , Motor Activity , Receptors, Dopamine D3ABSTRACT
We describe the cloning and characterization of a human 5-HT6 serotonin receptor. The open reading frame is interrupted by two introns in positions corresponding to the third cytoplasmic loop and the third extracellular loop. The human 5-HT6 cDNA encodes a 440-amino-acid polypeptide whose sequence diverges significantly from that published for the rat 5-HT6 receptor. Resequencing of the rat cDNA revealed a sequencing error producing a frame shift within the open reading frame. The human 5-HT6 amino acid sequence is 89% similar to the corrected rat sequence. The recombinant human 5-HT6 receptor is positively coupled to adenylyl cyclase and has pharmacological properties similar to the rat receptor with high affinity for several typical and atypical antipsychotics, including clozapine. The receptor is expressed in several human brain regions, most prominently in the caudate nucleus. The gene for the receptor maps to the human chromosome region 1p35-p36. This localization overlaps that established for the serotonin 5-HT1D alpha receptor, suggesting that these may be closely linked. Comparison of genomic and cDNA clones for the human 5-HT6 receptor also reveals an Rsal restriction fragment length polymorphism within the coding region.
Subject(s)
Brain Chemistry , Chromosomes, Human, Pair 1 , Genes , Receptors, Serotonin/genetics , Adenylyl Cyclases/metabolism , Amino Acid Sequence , Animals , Antipsychotic Agents/metabolism , Antipsychotic Agents/pharmacology , Base Sequence , Cell Line, Transformed , Chlorocebus aethiops , Chromosome Mapping , Cloning, Molecular , Clozapine/metabolism , Clozapine/pharmacology , DNA, Complementary/genetics , Humans , Kinetics , Molecular Sequence Data , Open Reading Frames , Protein Conformation , Rats , Receptors, Serotonin/drug effects , Receptors, Serotonin/metabolism , Recombinant Proteins/biosynthesis , Sequence Alignment , Sequence Homology, Nucleic Acid , Serotonin Agents , Signal Transduction , Species SpecificityABSTRACT
The serotonin receptor subtype 6, which raises intracellular cyclic AMP via stimulatory G-proteins, has recently been cloned and characterized. To determine the distribution of serotonin subtype 6 messenger RNA, in situ hybridization was performed in coronal sections of rat brain. 35S-labeled riboprobe, complementary to the 5' non-coding region of the serotonin subtype 6 messenger RNA, and a 33P-labeled riboprobe complementary to its 3' non-coding region, were used for hybridization. Serotonin subtype 6 receptor message was found in serotonin projection fields, rather than regions of serotonin-containing cell bodies, suggesting that the receptor is mainly postsynaptic. Hybridization signal was highest in olfactory tubercle, as well as prominent in the striatum, nucleus accumbens, dentate gyrus, and CA1, CA2 and CA3 of the hippocampus. Less intense hybridization was observed in cerebellum, some diencephalic nuclei, the amygdala, and layers 2, 3, 4 and 6 of the cortex. This pattern of hybridization was observed with both probes, but not when sense transcripts were used. Because the serotonin subtype 6 receptor has a high affinity for the atypical antipsychotic clozapine, and because striatum and nucleus accumbens are proposed sites of antipsychotic drug effects, the possibility is raised that this receptor may play an important role in mediating the effects of the atypical antipsychotic agents.
Subject(s)
Brain/physiology , RNA, Messenger/genetics , Receptors, Serotonin/physiology , Animals , Autoradiography , Basal Ganglia , Cerebral Cortex/physiology , In Situ Hybridization , Male , Rats , Rats, Sprague-Dawley , Receptors, Serotonin/classificationABSTRACT
A cDNA clone encoding a novel G protein-linked receptor was isolated from a rat cerebral cortex cDNA library using a polymerase chain reaction-amplified cDNA fragment as a probe. This 2.4-kb clone encodes a 367 amino acid protein with seven putative transmembrane spanning domains. The protein is highly homologous to the cloned micro, delta, and kappa opioid receptors and shares with them structural features such as three glycosylation sites in the amino terminus, a cyclic AMP-dependent kinase phosphorylation site in the third cytoplasmic loop, an aspartic acid residue in the second transmembrane domain, and a palmitoylation site on the intracellular carboxy terminus. The receptor is also homologous with members of the somatostatin receptor family, yet it binds neither opiate nor somatostatin ligands. Northern blot analysis reveals two transcripts of 3.2 and 7.6 kb that are predominantly expressed in the cerebral cortex and hypothalamus. In situ hybridization analysis also shows a high abundance of mRNA in the cerebral cortex, hippocampus, amygdala, hypothalamus, thalamus, and dorsal raphe nuclei. It is suggested that the endogenous ligand for this receptor may represent a novel neuropeptide that may be closely related to the opiate peptide family.
Subject(s)
DNA/analysis , GTP-Binding Proteins/genetics , Receptors, Cell Surface/genetics , Receptors, Opioid/genetics , Amino Acid Sequence , Animals , Base Sequence , Blotting, Northern , Brain Chemistry , CHO Cells , Cell Line , Cerebral Cortex/chemistry , Cerebral Cortex/ultrastructure , Cloning, Molecular , Cricetinae , DNA/genetics , GTP-Binding Proteins/analysis , Hippocampus/chemistry , Hippocampus/ultrastructure , In Situ Hybridization , Molecular Sequence Data , Polymerase Chain Reaction , RNA, Messenger/analysis , RNA, Messenger/genetics , Rats , Receptors, Cell Surface/analysis , Receptors, Opioid/analysis , Receptors, Opioid, delta/analysis , Receptors, Opioid, delta/genetics , Receptors, Opioid, kappa/analysis , Receptors, Opioid, kappa/genetics , Receptors, Opioid, mu/analysis , Receptors, Opioid, mu/genetics , Sequence Homology, Amino Acid , Sequence Homology, Nucleic Acid , TransfectionABSTRACT
Of the five known dopamine receptors, D1A and D2 represent the major subtypes expressed in the striatum of the adult brain. Within the striatum, these two subtypes are differentially distributed in the two main neuronal populations that provide direct and indirect pathways between the striatum and the output nuclei of the basal ganglia. Movement disorders, including Parkinson disease and various dystonias, are thought to result from imbalanced activity in these pathways. Dopamine regulates movement through its differential effects on D1A receptors expressed by direct output neurons and D2 receptors expressed by indirect output neurons. To further examine the interaction of D1A and D2 neuronal pathways in the striatum, we used homologous recombination to generate mutant mice lacking functional D1A receptors (D1A-/-). D1A-/- mutants are growth retarded and die shortly after weaning age unless their diet is supplemented with hydrated food. With such treatment the mice gain weight and survive to adulthood. Neurologically, D1A-/- mice exhibit normal coordination and locomotion, although they display a significant decrease in rearing behavior. Examination of the striatum revealed changes associated with the altered phenotype of these mutants. D1A receptor binding was absent in striatal sections from D1A-/- mice. Striatal neurons normally expressing functional D1A receptors are formed and persist in adult homozygous mutants. Moreover, substance P mRNA, which is colocalized specifically in striatal neurons with D1A receptors, is expressed at a reduced level. In contrast, levels of enkephalin mRNA, which is expressed in striatal neurons with D2 receptors, are unaffected. These findings show that D1A-/- mice exhibit selective functional alterations in the striatal neurons giving rise to the direct striatal output pathway.
Subject(s)
Corpus Striatum/physiology , Receptors, Dopamine/deficiency , Animals , Base Sequence , Gene Expression , Growth Disorders/genetics , In Situ Hybridization , Mice , Mice, Knockout , Molecular Sequence Data , Oligonucleotide Probes/chemistry , RNA, Messenger/genetics , Receptors, Dopamine/physiology , Restriction MappingABSTRACT
To investigate and compare the regulatory properties of the two isoforms of the D2 dopamine receptor, we have stably expressed their cDNAs in Chinese hamster ovary (CHO) cells. Cell lines were selected that express similar levels of [3H]methylspiperone-binding activity. Both isoforms mediate a dose-dependent and pharmacologically specific inhibition of adenylyl cyclase activity in both intact cell and membrane preparations. Pretreatment of both D2L and D2S receptor-expressing cells with 100 microM dopamine produces a approximately 5-fold shift (to lower affinity) in the EC50 for dopamine inhibition of cAMP accumulation, with a 25-30% decrease in the maximum response. Dopamine treatment also results in a approximately 25% decrease in the maximum receptor binding activity of the D2S receptor-expressing cells. In contrast, the D2L receptors are up-regulated by about 2-fold in response to dopamine exposure. This difference in response between the D2S and D2L receptors is not cell line specific, inasmuch as other CHO clones expressing these isoforms show identical responses. The dopamine-induced up-regulation of D2L receptor binding is time dependent, reaching maximal levels after 10 hr (t1/2 = 2 hr). Upon removal of dopamine, the receptor binding activity returns to control levels within 20 hr. The adenylyl cyclase desensitization response is also time dependent but exhibits a slower time course (t1/2 = 5 hr) than the receptor up-regulation. Both regulatory responses are induced in a dose-dependent fashion by dopamine, albeit with different potencies (up-regulation EC50 = 100 nM, desensitization EC50 = 2 microM). These regulatory effects are pharmacologically specific, being mimicked by D2-selective agonists but not agonists of other receptor subtypes. The dopamine-induced receptor up-regulation is blocked by prior treatment of the cells with pertussis toxin and is not mimicked by cAMP analogs. Conversely, elevation of intracellular cAMP levels results in down-regulation of the D2L receptor activity. To test whether protein synthesis is required for the D2L receptor up-regulation, cycloheximide was used to block mRNA translation. This was found to completely inhibit the up-regulation of D2L binding activity; however, there was no effect on the desensitization of the adenylyl cyclase response. RNA dot-blot analyses indicate that dopamine treatment is associated with a sustained 2-fold increase in the steady state levels of D2L mRNA, whereas D2S mRNA is transiently increased by only 50%.(ABSTRACT TRUNCATED AT 400 WORDS)