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

With a view toward improving the neural bioavailability of administered dopaminergic compounds, including dopamine, synthetic efforts have been directed toward enhancing the brain bioavailability of these compounds by accessing cellular sugar transport systems with stereoselective dopaminergic drugs. While synthesis and chemistry of the resultant class of compounds has recently been described in US Patent No. 6,548,484, the associated biologic properties have not previously been reported. One member of this new class, IPX-750, is a pro-drug dopamine-gluconamine designed to retain stereospecificity of binding at: glucose transporters (GLUT 1/GLUT 3 and intestinal Na+/glucose co-transporters SGLT1), dopamine transporter (DAT); and, dopaminergic receptors of the D1/D2 families. Designed to be cleavable by tissue amidases, results reported here show that intact IPX-750 pro-drug retains dopaminergic agonist binding and biologic activities both in vitro and in vivo. IPX-750, like dopamine, exhibited predominant D5/D1 binding specificity with lower binding activity at D2. As expected, binding was highly stereo-specific, ie, IPX-760, a benzamide differing in just a hydrogen atom and keto oxygen from IPX-750, bound with 6-fold lower activity at D5. In cell culture, activation resulted from binding of IPX-750 at D1 or D5 in transfected cells was measured by increased intracellular cAMP. Interestingly, considering prior reported in vitro toxicity of dopamine oxidized and metabolic product dopamine, no evidence of in vitro toxicity was observed at up to 72 hrs in cell cultures at the EC50 of IPX-750 for increasing intracellular cAMP. IPX-750 was evaluated in the Parkinson's disease animal models, including MPTP mouse model, the 6-hydroxydopamine (6-OHDA) rat model and the Nurr1(+/−) knockout mouse model. In MPTP-lesioned and Nurr1+/ − knockout mice, IPX-750 significantly increased Rota-rod time. In 6-OHDA-lesioned rats, IPX-750 significantly decreased apomorphine (APO)-induced rotation. Worthy of note, after cessation of IPX-750 treatments the anti-parkinsonian activity in MPTP-lesioned and Nurr1+/ − mice required about 2 weeks to washout, suggesting a possible biologic reservoir of drug. In addition, after eight weeks of twice daily administration of 20 mg/kg IPX-750, mice did not show statistical difference in the total number of TH-positive neurons in substantia nigra (SN). These combined results suggest (i) that stereo-specific glycoconjugation may be an effective method to improve penetrability of drugs through the blood brain barrier; (ii) treatment with bioavailable IPX-750 in vitro did not show evidence for neurotoxicity; and, (iii) IPX-750 possesses dopaminergic properties and exerts anti-parkinsonian effects in three different PD rodent models, suggesting therapeutic potential for this new class of drugs in treating dopamine deficiency diseases.

Alpha-synuclein and its missense mutants (A30P, A53T) have been linked to the genesis of idiopathic and rare familial forms of Parkinson's disease, respectively. Here we show that, similar to the wild-type alpha-synuclein, the A30P mutant forms a strong complex with the human dopamine transporter (hDAT), through direct protein:protein interactions between the nonamyloid beta component (NAC) domain of the A30P mutant and the last 22 aminoacyl residues of the carboxy-terminal tail of hDAT. The A30P mutant negatively modulates hDAT functional activity and to a greater extent than wild-type alpha-synuclein, with reduced uptake of extracellular dopamine and dopamine-mediated, hDAT-dependent cytotoxicity. By contrast, the A53T mutant neither forms a strong protein:protein complex with hDAT nor modulates dopamine uptake by hDAT, and dopamine-mediated, hDAT-dependent cytotoxicity is higher than with either wild-type or the A30P variant of alpha-synuclein, but not significantly different from that of cells expressing hDAT alone. Confocal microscopy shows substantial overlap in colocalization of all three alpha-synuclein variants with hDAT, with only minor differences. Although the complex formation with hDAT occurs through the NAC domain of the alpha-synuclein variants, it is the familial Parkinson's disease-linked missense mutations present in the amino-terminal lipid binding domain of the alpha-synuclein variants that dictate the extent of the regulation of hDAT function. These studies highlight previously unknown properties of the A30P and the A53T mutants of alpha-synuclein with respect to the modulation of hDAT activity and/or regulation, and its subsequent functional outcome, which are uniquely distinct.

Initial classification of dopamine (DA) receptors into D1 and D2 subtypes on the basis of stimulatory and no/inhibitory linkage to adenylyl cyclase (AC), respectively (Spano et al 1978; Kebabian and Calne 1979), endured in substance for approximately a decade until the molecular cloning of D1 and D2 receptors provided additional criteria for distinguishing these two receptors in terms of genomic structure/localization, primary structure and mRNA tissue distribution profile. Thereafter, primarily during the early 1990s, further molecular cloning studies revealed the mammalian DA receptor family to be yet more heterogeneous (see Missale et al. 1998; Neve and Neve 1997; Niznik 1994): in particular, cloning both of primate D1 (rodent homologue D1A ) and of primate D5 (rodent homologue D1B ) receptors indicated the original designation of D1 to encompass a family of D1-like receptors whose properties are t he focus of this chapter, in juxtaposition with a family of D2-like receptors (D2L/S, D3, D4) whose properties are the focus of subsequent chapters.

Metallothionein III (MT-III) is a functionally distinct member of the metallothionein family that displays neuroinhibitory activity and is involved in the repair of neuronal damage. Altered expression levels of MT-III have been observed in Alzheimer's disease (AD) which has led to suggestions that it could be a mitigating factor in AD-related neuronal dysfunction. However, conflicting results have been reported on this issue which may be due to methodological differences and/or sampling size. In the current study, we have assessed MT-III expression in a large number of AD cases through the quantification of mRNA as well as by immunohistochemistry and Western blotting using an MT-III specific antibody. The results of this comprehensive study indicate that the mononucleosome DNA encoding MT-III is occluded preventing transcription and that message levels are reduced by ∼30%. In addition, protein levels were specifically decreased by ∼55% in temporal cortex. These data support the conclusion that MT-III is significantly downregulated in AD and may contribute to the loss of its protective effects and/or repair functions that lead to an exacerbation of the pathogenic processes.

GABA A (γ-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 γ2(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.

The family of five dopamine receptors subtypes activate cellular effector systems through G proteins. Historically, dopamine receptors were thought to only stimulate or inhibit adenylyl cyclase, by coupling to either Gsα or Giα, respectively. Recent studies in transfected cells, reviewed here, have shown that multiple and highly diverse signaling pathways are activated by specific dopamine receptor subtypes. This multiplicity of signaling responses occurs through selective coupling to distinct G proteins and each of the receptors can interact with more than one G protein. Although some of the multiple coupling of dopamine receptors to different G proteins occurs from within the same family of G proteins, these receptors can also couple to G proteins belonging to different families. Such multiple interactions between receptors and G proteins elicits functionally distinct physiological effects which acts to enhance and subsequently suppress the original receptor response, and to activate apparently distinct signaling pathways. In the brain, where coexpression of functionally distinct receptors in heterogenous cells further adds to the complexity of dopamine signaling, minor alterations in receptor/G protein coupling states during either development or in adults, may underlie the imbalanced signaling seen in dopaminergic-linked diseases such as schizophrenia, Parkinson’s disease and attention deficit hyperactivity disorder.

Melatonin, a hormone principally produced and released by the pineal gland, has been shown to regulate a variety of biological functions including circadian rhythms, sleep-wake cycles and reproduction 1 , presumably through activating high-affinity G-protein-coupled receptors 2 , 3 , 4 , 5 . We report here that these subtypes can differentially modulate the function of type-A γ-aminobutyric acid (GABA A ) receptor, the principal neurotransmitter receptor mediating synaptic inhibition in the CNS 6 , 7 . This work demonstrates that melatonin, through activation of different receptor subtypes, can exert opposite effects on the same substrate, suggesting that receptor subtype is the primary molecular basis for the diversity of melatonin effects.

The activity of the dopamine transporter is an important mechanism for the maintenance of normal dopaminergic homeostasis by rapidly removing dopamine from the synaptic cleft. In kidney‐derived COS‐7, COS‐1 and HEK‐293 but not in other mammalian cell lines (CHO, Y1, Ltk−), we have characterized a putative functional dopamine transporter displaying a high affinity (K m∼250 nM) and a low capacity (∼0.1 pmol/105 cells/min) for [3H]dopamine uptake. Uptake displayed a pharmacological profile clearly indicative of the neuronal dopamine transporter. Estimated K i values of numerous substrates and inhibitors for the COS‐dopamine transporter and the cloned human neuronal transporter (human dopamine transporter) correlate well with the exception of a few notable compounds, including the endogenous neurotransmitter dopamine, the dopamine transporter inhibitor GBR 12,909 and the dopaminergic agonist apomorphine. As with native neuronal and cloned dopamine transporters, the uptake velocity was sodium‐sensitive and reduced by phorbol ester pre‐treatment. Two mRNA species of 3.8 and 4.0 kb in COS‐7 cells were revealed by Northern blot analysis similar in size to that seen in native neuronal tissue. A reverse‐transcribed PCR analysis confirmed the existence of a processed dopamine transporter. However, no immunoreactive proteins of expected dopamine transporter molecular size or [3H]WIN 35,428 binding activity were detected. A partial cDNA of ∼1.3 kb, isolated from a COS‐1 cDNA library and encoding transmembrane domains 1–6, displayed a deduced amino acid sequence homology of ∼96% to the human dopamine transporter. Taken together, the data suggest the existence of a non‐neuronal endogenous high affinity dopamine uptake system sharing strong functional and molecular homology to that of the cloned neuronal dopamine transporter.

The existence of dopamine D1C and D1D receptors in Xenopus and chicken, respectively, challenged the established duality (D1A and D1B) of the dopamine D1 receptor class in vertebrates. To ascertain the molecular diversity of this gene family in early diverging vertebrates, we isolated four receptor-encoding sequences from the European eel Anguilla anguilla. Molecular phylogeny assigned two receptor sequences (D1A1 and D1A2) to the D1A subtype, and a third receptor to the D1B subtype. Additional sequence was orthologous to the Xenopus D1C receptor and to several other previously unclassified fish D1-like receptors. When expressed in COS-7 cells, eel D1A and D1B receptors display affinity profiles for dopaminergic ligands similar to those of other known vertebrate homologues. The D1C receptor exhibits pharmacological characteristics virtually identical to its Xenopus homologue. Functionally, while all eel D1 receptors stimulate adenylate cyclase, the eel D1B receptor exhibits greater constitutive activity than either D1A or D1C receptors. Semiquantitative reverse transcription-polymerase chain reaction reveals the differential distribution of D1A1, D1A2, D1B, and D1C receptor mRNA within the hypothalamic-pituitary axis of the eel brain. Taken together, these data suggest that the D1A, D1B, and D1C receptors arose prior to the evolutionary divergence of fish and tetrapods and exhibit molecular, pharmacological, and functional attributes that unambiguously allow for their classification as distinct D1 receptor subtypes in the vertebrate phylum.

Blockade of N-methyl-D-aspartate (NMDA) receptors by the specific antagonists dizocilpine and (+/-)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid on human neuroblastoma SH-SY5Y cells expressing human D21 receptors resulted in a significant increase in the density of D2L receptors. In order to understand the mechanism of dopamine D2L receptor induction following NMDA receptor blockade we used specific protein tyrosine kinase and phosphatase inhibitors to demonstrate their involvement in this interaction. The induction of dopamine D2L receptor was measured by radioreceptor binding assay. The density of the dopamine D2L receptor was increased to 109% by the inhibition of protein tyrosine kinase and prevented by the inhibition of phosphatase 1 or 2A. Inactivation of NMDA receptors might effect the phosphorylation-dephosphorylation states of the regulatory proteins and lead to the induction of the D2L receptor gene.

Abstract: To understand the mechanism of interaction of the dopamine D2L receptors with NMDA receptors, we have developed a model by transfecting human neuroblastoma SH‐SY5Y cells with the human dopamine D2L receptor gene. In vitro blockade of NMDA receptors by the specific antagonists MK‐801 and (±)‐3‐(2‐carboxypiperazin‐4‐yl)‐propyl‐1‐phosphonic acid (CPP) on human neuroblastoma SH‐SY5Y cells expressing human dopamine D2L receptors resulted in a significant increase in the density of D2L receptors without a significant change in receptor affinity. Moreover, the dopamine receptor mRNA level increased by ∼50% by the blockade of NMDA with MK‐801. These results suggest a possible interaction of NMDA and dopamine D2L receptors in neuroblastoma SH‐SY5Y cells. This system would serve as an excellent model to study the molecular mechanisms involved in the interaction of these two receptors.

Previously, we have reported the cloning and characterization of the 5'-flanking region of the human dopamine D5 receptor encoding gene (D5) and that the major transactivation domain was 119-182 bp upstream of the transcriptional start site [Beischlag, TV. et al., Biochemistry, 34 (1995) 5960-5970]. Within this region existed a small dinucleotide repeat termed (TC)(13) In this report, we describe the screening of genomic DNAs from 18 unrelated individuals by single-strand conformation polymorphism (SSCP) analysis. SSCP analysis revealed the existence of two additional alleles, termed (TC)(12) and (TC)(14). Neither form significantly altered D5 promoter-mediated luciferase activity when compared to that of the wild-type control, suggesting that small differences in the number of dinucleotide repeats are not likely of any functional consequence for D5 transactivation.

Three genomic clones encoding dopamine D1-like receptors were isolated from the avian species Gallus domesticus. Two of these genes encode proteins of 451 and 488 amino acids, which, based on deduced amino acid sequence identity and homology of exhibited pharmacological profiles, appear to be species homologs of mammalian and vertebrate D1/D1A and D5/D1B receptors, respectively. The third genomic clone, termed D1D, encodes a protein of 445 amino acids displaying a deduced amino acid sequence identity within putative transmembrane domains of 75% to mammalian D1/D1A and 77% to D5/D1B receptors with overall sequence homologies of only 49% and 46%, respectively. Membranes from COS-7 cells transfected with D1D DNA bound [3H]SCH-23390 in a saturable manner with high affinity (approximately 300 pM) and with a pharmacological profile clearly indicative of a dopamine D1-like receptor. The D1D receptor exhibited affinities for 6,7-dihydroxy-2-aminotetralin and dopamine 10-fold higher than D1/D1A receptors, characteristic of the D5/D1B receptor subfamily. In contrast, the D1D receptor bound dopaminergic agents, such as SKF-38393, apomorphine, pergolide, and lisuride, with affinities 10-fold higher than other cloned mammalian or vertebrate D1A/D1B receptor subtypes, while both clozapine and haloperidol displayed considerably lower affinity for the D1D receptor. Based on the low overall amino acid sequence identity (54%) and unique pharmacological profile, the avian dopamine D1D receptor does not appear to be a species homolog of the recently cloned vertebrate D1C receptor (Sugamori, K.S., Demchyshyn, L. L., Chung, M., and Niznik, H. B. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 10536-10540). As with all cloned mammalian and vertebrate D1-like receptors, the D1D receptor stimulates adenylate cyclase activity in the presence of dopamine or SKF-82526. Northern blot analysis reveals the selective expression of both avian D1D and D1A receptor mRNAs only in brain with the D1B receptor more widely distributed and localized in tissues such as brain, kidney, and spleen. The isolation of four distinct vertebrate dopamine D1 receptor subtypes suggests the existence of additional mammalian D1 like receptor genes that may account for the observed pharmacological and biochemical multiplicity of dopamine D1-like receptor mediated events.

The diverse biological effects of somatostatin (SST) are mediated through a family of G protein coupled receptors of which 5 members have been recently identified by molecular cloning. This review focuses on the molecular biology, pharmacology, expression and function of these receptors with particular emphasis on the human (h) homologs. hSSTRs are encoded by a family of 5 genes which map to separate chromosomes and which, with one exception, are intronless. SSTR2 gives rise to spliced variants, SSTR2A and 2B. hSSTR 1–4 display weak selectivity for SST-14 binding whereas hSSTRS is SST-28 selective. Based on structural similarity and reactivity for octapeptide and hexapeptide SST analogs, hSSTR2,3 and 5 belong to a similar SSTR subclass. hSSTRl and 4 react poorly with these analogs and belong to a separate subclass. All 5 hSSTRs are functionally coupled to inhibition of adenylyl cyclase via pertussis toxin sensitive GTP binding proteins. Some of the subtypes are also coupled to tyrosine phosphatase (SSTR1,2), Ca2+ channels (SSTR2), Na+/H+ exchanger (SSTR1), PLA-2 (SSTR4), and MAP kinase (SSTR4). mRNA for SSTR1-5 is widely expressed in brain and peripheral organs and displays an overlapping but characteristic pattern that is subtypeselective and tissue- and species-specific. Pituitary and islet tumors express several SSTR genes suggesting that multiple SSTR subtypes are coexpressed in the same cell. Structure-function studies indicate that the core residues in SST-14 ligand Phe6-Phe11 dock within a ligand binding pocket located in TMDs 3–7 which is lined by hydrophobic and charged amino acid residues.

Part 1 Physiology and Pharmacology of Dopamine Receptors: Dopamine Receptors: Radioligands for Pharmacological and Biochemical Characterization The Neuromodulatory Role of Dopamine in the Teleost Retina Coupling of Dopamine Receptors to Ionic Channels in Excitable Tissues Peripheral Dopamine Receptors Invertebrate Dopamine Receptors. Part 2 Molecular Biology of Dopamine Receptors: Dopamine D1a and D1b Receptors The Dopamine-D2 Receptor The Dopamine-D3 Receptor The Dopamine-D4 Receptor Dopamine Receptor Genes: Promoters and Transcriptional Activation The Evolution of the Dopamine Receptor Gene Family. Part 3 Dopamine Receptors: Structure and Function Molecular Modelling of Dopamine Receptors Dopamine-D1 Receptor: Structure and Function Structure Function Analysis of Dopamine-D2 Receptors by In Vitro Mutagenesis Functional Distinctions of D2Long and D2Short Receptors. Part 4 Anatomical Distribution of the Dopamine Receptor System: distribution of Dopamine D1- and D2-Like Dopamine Receptors in Human Brain Immunocytochemical Localization of D1 and D2 Receptors in Rat Brain Neuroanatomical Distribution of Dopamine Receptor Messenger RNAs. Part 5 Dopamine Receptor/Gene Regulation: Dopamine Receptor Regulation of Immediate-Early Genes in the Basal Ganglia Dopamine Receptor Gene Expression: Effects of Neuroleptics, Denervation, and Development Regulation of D2 Receptor Expression and Function by Epidermal Growth Factor in GH-3 Cells Dopaminergic Signalling and Regulation of Pituitary Hormone Genes The Chakragati Mouse: A Model for Brain Dopaminergic Dysfunction Levels of Functional Interaction Between D1-Like and D2-Like Dopamine Receptor Systems. Part 6 Dopamine Receptors and Molecular Psychiatry: Dopamine Receptors in Schizophrenia Dopamine Receptor and Neurotransmitter Imaging in Psychiatry Illness Molecular Genetics of the Dopamine Receptor Genes and Schizophrenia Dopamine Receptors in Neurological Disease The Dopamine Transporter Molecular Biology of Dopamine Transporters.

We have recently cloned a novel human 5-HT1D receptor subtype termed 5-HT1Dβ. CHO K1 cells expressing the human serotonin 5-HT1Dβ receptor were assayed to determine the second messenger system of this receptor. Cyclic AMP radioimmunoassays revealed that the 5-HT1Dβ receptor is negatively coupled to adenylate cyclase in this cell system. A maximum of 50% inhibition of forskolin stimulated cAMP production was obtained with 5-HT1 receptor agonists which was blocked by the non-selective 5-HT receptor antagonist methiothepin (pKB = 100 nM). The novel anti-migraine drug sumatriptan, a putative 5-HT1D selective compound, acted as an agonist at the 5-HT1Dβ receptor. Most notably metergoline, a putative 5-HT1 receptor antagonist, did not block the effects of 5-HT and was found to be acting as a full agonist at the 5-HT1Dβ receptor. The ability of metergoline to act as an agonist at the 5-HT1Dβ receptor may explain why it does not inhibit 5-HT and sumatriptan induced contraction of dog saphenous vein and other large conducting arteries. These results suggest that the 5-HT1Dβ receptor may be the site of action of sumatriptan in preventing migraine, and that metergoline's actions on the dog saphenous vein are not contradictory to that hypothesis, as previously reported.