Molecular biology of GABAA receptors

The major type of receptor for the inhibitory neurotransmitter gamma-aminobutyric acid (GABA), called the GABAA receptor, is a member of a gene superfamily of ligand-gated ion channels. This receptor is a hetero-oligomeric protein composed of several distinct polypeptide types (alpha, beta, gamma, and delta). Molecular cloning of these polypeptides reveals that they show 20-40% identity with each other, and 10-20% identity with polypeptides of the nicotinic acetylcholine receptors and strychnine-sensitive glycine receptor. Each polypeptide type is also represented by a family of genes whose members have 60-80% amino acid sequence identity. Regions of conserved and variable amino acid sequence suggest structural and functional domains within each polypeptide. All of the polypeptides when expressed in heterologous cells produce GABA-activated chloride channels, and the different subtypes express different pharmacological properties. The distributions of mRNAs for the different GABAA receptor polypeptides and their subtypes show significant brain regional variation consistent with pharmacological and biochemical evidence for receptor heterogeneity. Subpopulations of GABAA receptors with different cellular and regional locations show differential sensitivity to GABA, to modulators like steroids, to physiological regulation, to disease processes, and to pharmacological manipulation by drugs such as benzodiazepines. The properties of the different subpopulations of GABAA receptors are determined by which one or more of the different polypeptides and their subtypes are expressed in a given cell to produce a variety of different oligomeric protein structures. Molecular cloning techniques have produced rapid advances in understanding the GABAA receptor protein family.     

Critical role of peptidic toxins in the functional and structural analysis of nicotinic acetylcholine receptors

Animal toxins which interact on various receptors and channels have been often used in the studies of the functional roles of these targets. Nicotinic toxins have been purified from snake and cone venoms and are characterized by high affinity and various selectivity of interactions on the different nicotinic receptors subtypes. Since 30 years they have been used as molecular probes to identify, localize and purify these receptors. Furthermore, they have played a crucial role in the better understanding of their functional properties and have been useful in their structural studies. These peptidic toxins could be chemically synthetized or recombinantly expressed and nonnatural residues could be introduced in their sequences in order to delineate their functional interaction sites. The structural modelisation of toxin-nAChR interaction allows us to understand the antagonistic property of these toxins and open the way to the design of engineered ligands with predetermined specificity, useful as pharmacological tools or therapeutic agents in the numerous diseases involving this receptor family.     

Evolution and cell biology of dopamine receptors in vertebrates

Dopamine, one of main modulatory neurotransmitters of the nervous system acts on target cells through two classes of G protein-coupled receptors, D1 and D2. The two dopamine receptor classes display different structures, interact with different regulatory partners (including heterotrimeric G proteins) and, accordingly, have independent evolutionary origins. In vertebrates, each of these receptor classes comprises several subtypes, generated by two steps of gene duplications, early in vertebrate evolution. In the D1 receptor class, the D1A, D1B, D1C and D1D subtypes, and in the D2 class, the D2, D3 and D4 receptor subtypes have been conserved in most vertebrate groups. This conservation has been driven by the acquisition, by each receptor subtype, of a small number of specific properties, which were selected for adaptive purpose in vertebrates. Among these properties, affinity for dopamine, the natural ligand, intrinsic receptor activity, and agonist-induced desensitization clearly distinguish the receptor subtypes. In addition, each dopamine receptor subtype is addressed to a specific location within neuronal networks, although detailed information is lacking for several receptor subtypes. Receptors localization at diverse subcellular places in neurons may also differ from one subtype to another, resulting in different ways of regulating cell signalisation. One challenge for future research on dopamine and its receptors would be to identify the nature of the protein partners and the molecular mechanisms involved in localizing receptors to the neuronal plasma membrane. In this respect, the evolutionary approach we have undertaken suggests that, due to gene duplications, a reasonable degree of freedom exists in the tight organisation of dopamine receptors in neurons. This "evolvability" of dopamine systems has been instrumental to adapt the vertebrate species to nearly all the possible environments.     

Cloning and characterization of a Caenorhabditis elegans D2-like dopamine receptor

The neurotransmitter dopamine plays an important role in the regulation of behavior in both vertebrates and invertebrates. In mammals, dopamine binds and activates two classes of dopamine receptors, D1-like and D2-like receptors. However, D2-like dopamine receptors in Caenorhabditis elegans have not yet been characterized. We have cloned a cDNA encoding a putative C. elegans D2-like dopamine receptor. The deduced amino acid sequence of the cloned cDNA shows higher sequence similarities to vertebrate D2-like dopamine receptors than to D1-like receptors. Two splice variants that differ in the length of their predicted third intracellular loops were identified. The receptor heterologously expressed in cultured cells showed high affinity binding to [125I]iodo-lysergic acid diethylamide. Dopamine showed the highest affinity for this receptor among several amine neurotransmitters tested. Activation of the heterologously expressed receptor led to the inhibition of cyclic AMP production, confirming that this receptor has the functional property of a D2-like receptor. We have also analyzed the expression pattern of this receptor and found that the receptor is expressed in several neurons including all the dopaminergic neurons in C. elegans.     

Synthesis and characterization of selective dopamine D2 receptor antagonists

A series of indole compounds have been prepared and evaluated for affinity at D2-like dopamine receptors using stably transfected HEK cells expressing human D2, D3, or D4 dopamine receptors. These compounds share structural elements with the classical D2-like dopamine receptor antagonists, haloperidol, N-methylspiperone, and benperidol. The compounds that share structural elements with N-methylspiperone and benperidol bind non-selectively to the D2 and D3 dopamine receptor subtypes. However, several of the compounds structurally similar to haloperidol were found to (a) bind to the human D2 receptor subtype with nanomolar affinity, (b) be 10- to 100-fold selective for the human D2 receptor compared to the human D3 receptor, and (c) bind with low affinity to the human D4 dopamine receptor subtype. Binding at sigma (sigma) receptor subtypes, sigma1 and sigma2, were also examined and it was found that the position of the methoxy group on the indole was pivotal in both (a) D2 versus D3 receptor selectivity and (b) affinity at sigma1 receptors. Adenylyl cyclase studies indicate that our indole compounds with the greatest D2 receptor selectivity are neutral antagonists at human D2 dopamine receptor subtypes. With stably transfected HEK cells expressing human D2 (hD2-HEK), these compounds (a) have no intrinsic activity and (b) attenuated quinpirole inhibition of adenylyl cyclase. The D2 receptor selective compounds that have been identified represent unique pharmacological tools that have potential for use in studies on the relative contribution of the D2 dopamine receptor subtypes in physiological and behavioral situations where D2-like dopaminergic receptor involvement is indicated.     

Functional Differentiation of Multiple Dopamine D1-Like Receptors by NNC 01-0012

Abstract: Although members of the multiple vertebrate/mammalian dopamine D1 receptor gene family can be selectively classified on the basis of their molecular/phylogenetic, structural, and tissue distribution profiles, no subtype-specific discriminating agents have yet been identified that can functionally differentiate these receptors. To define distinct pharmacological/functional attributes of multiple D1-like receptors, we analyzed the ligand binding profiles, affinity, and functional activity of 12 novel NNC compounds at mammalian/vertebrate D1/D1A and D5/D1B, as well as vertebrate D1C/D1D, dopamine receptors transiently expressed in COS-7 cells. Of all the compounds tested, only NNC 01-0012 displayed preferential selectivity for vertebrate D1C receptors, inhibiting [³H]SCH-23390 binding with an estimated affinity (∼0.6 n M ) 20-fold higher than either mammalian/vertebrate D1/D1A or D5/D1B receptors or the D1D receptor. Functionally, NNC 01-0012 is a potent antagonist at D1C receptors, inhibiting to basal levels dopamine (10 µ M )-stimulated adenylyl cyclase activity. In contrast, NNC 01-0012 (10 µ M ) exhibits weak antagonist activity at D1A receptors, inhibiting only 60% of maximal cyclic AMP production by dopamine, while acting as a partial agonist at vertebrate D1B and D1D receptors, stimulating adenylyl cyclase activity by ∼33% relative to the full agonist dopamine (10 µ M ), an effect that was blocked by the selective D1 receptor antagonist NNC 22-0010. These data clearly suggest that the benzazepine NNC 01-0012, despite lacking the N -methyl residue in the R3 position, is a selective and potent D1C receptor antagonist. Moreover, the differential signal transduction properties exhibited by NNC 01-0012 at these receptor subtypes provide further evidence, at least in vertebrates, for the classification of the D1C receptor as a distinct D1 receptor subtype.     

Delineation of the conserved functional properties of D1A, D1B and D1C dopamine receptor subtypes in vertebrates

The three main subtypes of dopamine D(1) receptor (D(1A), D(1B) and D(1C)) subtypes found in most vertebrate groups were generated by two major steps of gene duplications, early in evolution. To identify the functional characteristics contributing to conservation of these paralogous D(1) receptors in vertebrates, the pharmacological and functional properties of fish (Anguilla anguilla), amphibian (Xenopus laevis) and human receptors were systematically analysed in transfected cells. The ligand-binding parameters appeared essentially similar for orthologous receptors, but differed significantly among the subtypes. The D(1A) receptors from the three species displayed low intrinsic activity and a fast rate of agonist-induced desensitization. All the orthologous D(1B) receptors exhibited a similar desensitization time-course, but with smaller amplitude of decrease than D(1A) receptors, in agreement with their higher basal activity. In contrast, D(1C) receptors, which do not exist in mammals, have low intrinsic activity and exhibit only weak, but rapid, agonist-induced desensitization, without any changes upon longer treatment with agonist. Thus, each of the three D(1) receptor subtypes are characterized by activation and desensitization properties, in a sequence-specific manner, which has been probably acquired early after gene duplications, and constrained their conservation during vertebrate evolution. These properties have been instrumental to adapt dopamine system to the physiology of the numerous neuronal networks and functions they control in the large and complex brains of vertebrates.     

Delineation of the structural basis for the activation properties of the dopamine D1 receptor subtypes.

To delineate the structural determinants involved in the constitutive activation of the D1 receptor subtypes, we have constructed chimeras between the D1A and D1B receptors. These chimeras harbored a cognate domain corresponding to transmembrane regions 6 and 7 as well as the third extracellular loop (EL3) and cytoplasmic tail, a domain referred herein to as the terminal receptor locus (TRL). A chimeric D1A receptor harboring the D1B-TRL (chimera 1) displays an increased affinity for dopamine that is indistinguishable from the wild-type D1B receptor. Likewise, a chimeric D1B receptor containing the D1A-TRL cassette (chimera 2) binds dopamine with a reduced affinity that is highly reminiscent of the dopamine affinity for the wild-type D1A receptor. Furthermore, we show that the agonist independent activity of chimera 1 is identical to the wild-type D1B receptor whereas the chimera 2 displays a low agonist independent activity that is indistinguishable from the wild-type D1A receptor. Dopamine potencies for the wild-type D1A and D1B receptor were recapitulated in cells expressing the chimera 2 or chimera 1, respectively. However, the differences observed in agonist-mediated maximal activation of adenylyl cyclase elicited by the D1A and D1B receptors remain unchanged in cells expressing the chimeric receptors. To gain further mechanistic insights into the structural determinants of the TRL involved in the activation properties of the D1 receptor subtypes, we have engineered two additional chimeric D1 receptors that contain the EL3 region of their respective cognate wild-type counterparts (hD1A-EL3B and hD1B-EL3A). In marked contrast to chimera 1 and 2, dopamine affinity and constitutive activation were partially modulated by the exchange of the EL3. Meanwhile, hD1A-EL3B and hD1B-EL3A mutant receptors display a full switch in the agonist-mediated maximal activation, which is reminiscent of their cognate wild-type counterparts. Overall, our studies suggest a fundamental role for the TRL in shaping the intramolecular interactions implicated in the constitutive activation and coupling properties of the dopamine D1 receptor subtypes.     

Amphioxus expresses both vertebrate-type and invertebrate-type dopamine D1 receptors

The cephalochordate amphioxus (Branchiostoma floridae) has recently been placed as the most basal of all the chordates, which makes it an ideal organism for studying the molecular basis of the evolutionary transition from invertebrates to vertebrates. The biogenic amine, dopamine regulates many aspects of motor control in both vertebrates and invertebrates, and in both cases, its receptors can be divided into two main groups (D1 and D2) based on sequence similarity, ligand affinity and effector coupling. A bioinformatic study shows that amphioxus has at least three dopamine D1-like receptor sequences. We have recently characterized one of these receptors, AmphiD1/β, which was found to have high levels of sequence similarity to both vertebrate D1 receptors and to β-adrenergic receptors, but functionally appeared to be a vertebrate-type dopamine D₁ receptor. Here, we report on the cloning of two further dopamine D₁ receptors (AmphiAmR1 and AmphiAmR2) from adult amphioxus cDNA libraries and their pharmacological characterisation subsequent to their expression in cell lines. AmphiAmR1 shows closer structural similarities to vertebrate D₁-like receptors but shows some pharmacological similarities to invertebrate “DOP1” dopamine D₁-like receptors. In contrast, AmphiAmR2 shows closer structural and pharmacological similarities to invertebrate “INDR”-like dopamine D₁-like receptors.     

Differential sensitivity of two insect GABA-gated chloride channels to dieldrin,fipronil and picrotoxinin

In the central nervous system of both vertebrates and invertebrates inhibitory neurotransmission is mainly achieved through activation of γ-aminobutyric acid (GABA) receptors. Extensive studies have established the structural and pharmacological properties of vertebrate GABA receptors. Although the vast majority of insect GABA-sensitive responses share some properties with vertebrate GABAA receptors, peculiar pharmacological properties of these receptors led us to think that several GABA-gated chloride channels are present in insects. We describe here the pharmacological properties of two GABA receptor subtypes coupled to a chloride channel on dorsal unpaired median (DUM) neurones of the adult male cockroach. Long applications of GABA induce a large biphasic hyperpolarization, consisting of an initial transient hyperpolarization followed by a slow phase of hyperpolarization that is not quickly desensitized. With GABA, the transient hyperpolarization is sensitive to picrotoxinin, fipronil and dieldrin whereas the slow response is insensitive to these insecticides.When GABA is replaced by muscimol and cis-4-aminocrotonic acid (CACA) a biphasic hyperpolarization consisting of an initial transient hyperpolarization followed by a sustained phase is evoked which is blocked by picrotoxinin and fipronil. Exposure to dieldrin decreases only the early phase of the muscimol and CACA-induced biphasic response, suggesting that two GABA-gated chloride channel receptor subtypes are present in DUM neurones. This study describes, for the first time, a dieldrin resistant component different to the dieldrin- and picrotoxinin-resistant receptor found in several insect species.     

Pharmacological characterization of mammalian D1 and D2 dopamine receptors expressed in Drosophila Schneider-2 cells

Mammalian D1 and D2 dopamine receptors were stably expressed in Drosophila Schneider-2 (S2) cells and screened for their pharmacological properties. Saturable, dose-dependent, high affinity binding of the D1-selective antagonist [3H]SCH-23390 was detected only in membranes from S2 cells induced to express rat dopamine D1 receptors, while saturable, dose-dependent, high affinity binding of the D2-selective antagonist [3H]methylspiperone was detected only in membranes from S2 cells induced to express rat dopamine D2 receptors. No specific binding of either radioligand could be detected in membranes isolated from uninduced or untransfected S2 cells. Both dopamine D1 and D2 receptor subtypes displayed the appropriate stereoselective binding of enantiomers of the nonselective antagonist butaclamol. Each receptor subtype also displayed the appropriate agonist stereoselectivities. The dopamine D1 receptor bound the (+)-enantiomer of the D1-selective agonist SKF38393 with higher affinity than the (-)-enantiomer, while the dopamine D2 receptor bound the (-)-enantiomer of the D2-selective agonist norpropylapomorphine with higher affinity than the (+)-enantiomer. At both receptor subtypes, dopamine binding was best characterized as occurring to a single low affinity site. In addition, the low affinity dopamine binding was also found to be insensitive to GTPgammaS and magnesium ions. Overall, the pharmacological profiles of mammalian dopamine D1 and D2 receptors expressed in Drosophila S2 cells is comparable to those observed for these same receptors when they are expressed in mammalian cell lines. A notable distinction is that there is no evidence for the coupling of insect G proteins to mammalian dopamine receptors. These results suggest that the S2 cell insect G system may provide a convenient source of pharmacologically active mammalian D1 and D2 dopamine receptors free of promiscuous G protein contaminants.     

Mannose-6-phosphate receptors (MPR 300 and 46) from the highly evolved invertebrate <Emphasis Type="Italic">Asterias rubens</Emphasis> (Echinodermate): biochemical and functional characterization of MPR 46 protein

Mammalian mannose 6-phosphate receptors (MPR 300 and 46) mediate transport of lysosomal enzymes to lysosomes. Recent studies established that the receptors are conserved throughout vertebrates. Although we purified the mollusc receptors and identified only a lysosomal enzyme receptor protein (LERP) in the Drosophila melanogaster, little is known about their structure and functional roles in the invertebrates. In the present study, we purified the putative receptors from the highly evolved invertebrate, starfish, cloned the cDNA for the MPR 46, and expressed it in mpr^(−/−) mouse embryonic fibroblast cells. Structural comparison of starfish receptor sequences with other vertebrate receptors gave valuable information on its extensive structural homology with the vertebrate MPR 46 proteins. The expressed protein efficiently sorts lysosomal enzymes within the cells establishing a functional role for this protein. This first report on the invertebrate MPR 46 further confirms the structural and functional conservation of the receptor not only in the vertebrates but also in the invertebrates.     

The dopamine D(2) receptor is expressed and sensitizes adenylyl cyclase activity in airway smooth muscle

Dopamine receptors are G protein-coupled receptors that are divided into two subgroups, "D(1)-like" receptors (D(1) and D(5)) that couple to the G(s) protein and "D(2)-like" receptors (D(2), D(3), and D(4)) that couple to G(i). Although inhaled dopamine has been reported to induce bronchodilation in patients with asthma, functional expression of dopamine receptor subtypes has never been described on airway smooth muscle (ASM) cells. Acute activation of G(i)-coupled receptors inhibits adenylyl cyclase activity and cAMP synthesis, which classically impairs ASM relaxation. In contrast, chronic activation of G(i)-coupled receptors produces a paradoxical enhancement of adenylyl cyclase activity referred to as heterologous sensitization. We questioned whether the dopamine D(2)-like receptor is expressed on ASM, whether it exhibits classical G(i)-coupling, and whether it modulates ASM function. We detected the mRNA encoding the dopamine D(2) receptor in total RNA isolated from native human ASM and from cultured human airway smooth muscle (HASM) cells. Immunoblots identified the dopamine D(2) receptor protein in both native human and guinea pig ASM and cultured HASM cells. The dopamine D(2) receptor protein was immunohistochemically localized to both human and guinea pig ASM. Acute activation of the dopamine D(2) receptor by quinpirole inhibited forskolin-stimulated adenylyl cyclase activity in HASM cells, which was blocked by the dopamine D(2) receptor antagonist L-741626. In contrast, the chronic pretreatment (1 h) with quinpirole potentiated forskolin-stimulated adenylyl cyclase activity, which was inhibited by L-741626, the phospholipase C inhibitor U73122, or the protein kinase C inhibitor GF109203X. Quinpirole also stimulated inositol phosphate synthesis, which was inhibited by L-741626 or U73122. Chronic pretreatment (1 h) of the guinea pig tracheal rings with quinpirole significantly potentiated forskolin-induced airway relaxation, which was inhibited by L-741626. These results demonstrate that functional dopamine D(2) receptors are expressed on ASM and could be a novel therapeutic target for the relaxation of ASM.     

Methamphetamine Increases Locomotion and Dopamine Transporter Activity in Dopamine D5 Receptor-Deficient Mice

Dopamine regulates the psychomotor stimulant activities of amphetamine-like substances in the brain. The effects of dopamine are mediated through five known dopamine receptor subtypes in mammals. The functional relevance of D5 dopamine receptors in the central nervous system is not well understood. To determine the functional relevance of D5 dopamine receptors, we created D5 dopamine receptor-deficient mice and then used these mice to assess the roles of D5 dopamine receptors in the behavioral response to methamphetamine. Interestingly, D5 dopamine receptor-deficient mice displayed increased ambulation in response to methamphetamine. Furthermore, dopamine transporter threonine phosphorylation levels, which regulate amphetamine-induced dopamine release, were elevated in D5 dopamine receptor-deficient mice. The increase in methamphetamine-induced locomotor activity was eliminated by pretreatment with the dopamine transporter blocker GBR12909. Taken together, these results suggest that dopamine transporter activity and threonine phosphorylation levels are regulated by D5 dopamine receptors.     

Evolution of Dopamine-Related Systems: Biosynthesis, Degradation and Receptors

The evolution of enzyme genes at the pathway level has attracted increasing attention in recent years. Most investigations have focused on microorganisms, plants and invertebrates but rarely on vertebrates. The dopamine pathway, which participates in almost every aspect of brain function, is an excellent candidate for study at the pathway level. Herein, we report data on the divergence of six dopamine metabolic enzyme genes (three anabolic, three catabolic enzymes) and five dopamine receptor genes across five mammals, namely Homo sapiens, Pan troglodytes, Macaca mulatta, Mus musculus, and Rattus norvegicus. For enzyme genes, our data confirm previous conclusion that the upstream genes have evolved more slowly than downstream genes. Moreover, we found that catabolic genes in the dopamine metabolic pathway have evolved faster than anabolic genes, and maximum likelihood analysis suggested that this difference in evolutionary rate may be explained by anabolic genes being more constrained during selection. For dopamine receptor genes, however, the broadly expressed genes have tended to evolve more slowly than the narrowly expressed genes; maximum likelihood analysis showed that the relatively rapid evolutionary rate of the narrowly expressed receptor genes was a consequence of relaxed selective constraints. Finally, our data imply that selective constraints on synonymous sites in enzyme genes are relaxed compared with those of receptor genes because of differences in their patterns of functional regulation.     

GABAA receptor channels: from subunits to functional entities.

GABAA receptor channels mediate postsynaptic inhibition. The functional diversity of these receptors rests on differences in subunit composition and on a large repertoire of subunits. Subunit expression patterns in the brain have been found to predict in vivo compositions of GABAA receptors. In addition, molecular determinants underlying the differential binding properties of allosteric ligands to receptor subtypes have been identified.     

Molecular Approaches to Receptors as Targets for Drug Discovery

Abstract

The cloning of a great number of receptors and channels has revealed that many of these targets for drug discovery can be grouped into superfamilies based on sequence and structural similarities. This review presents an overview of how molecular biological approaches have revealed a plethora of receptor subtypes, led to new definitions of subtypes and isoforms, and played a role in the development of highly selective drugs. Moreover, the diversity of subtypes has molded current views of the structure and function of receptor families. Practical difficulties and limitations inherent in the characterization of the ligand binding and signaling properties of expressed recombinant receptors are discussed. The importance of evaluating drug-receptor interactions that differ with temporally transient and distinct receptor conformational states is emphasized. Structural motifs and signal transduction features are presented for the following major receptor superfamilies: ligand-gated ion channel, voltage-dependent ion channel, G-protein coupled, receptor tyrosine-kinase, receptor protein tyrosine-phosphatase, cytokine and nuclear hormone. In addition, a prototypic receptor is analyzed to illustrate functional properties of a given family. The review concludes with a discussion of future directions in receptor research that will impact drug discovery, with a specific focus on orphan receptors as targets for drug discovery. Methods for classifying orphan receptors based upon homologies with members of existing superfamilies are presented together with molecular approaches to the greater challenge of defining their physiological roles. Besides revealing new orphan receptors, the human genome sequencing project will result in the identification of an abundance of novel receptors that will be molecular targets for the development of highly selective drugs. These findings will spur the discovery and development of an exciting new generation of receptor-subtype specific drugs with enhanced therapeutic specificity.     

Pharmacological and signalling properties of a D2-like dopamine receptor (Dop3) in Tribolium castaneum

Dopamine is an important neurotransmitter in the central nervous system of vertebrates and invertebrates. Despite their evolutionary distance, striking parallels exist between deuterostomian and protostomian dopaminergic systems. In both, signalling is achieved via a complement of functionally distinct dopamine receptors. In this study, we investigated the sequence, pharmacology and tissue distribution of a D2-like dopamine receptor from the red flour beetle Tribolium castaneum (TricaDop3) and compared it with related G protein-coupled receptors in other invertebrate species.The TricaDop3 receptor-encoding cDNA shows considerable sequence similarity with members of the Dop3 receptor class. Real time qRT-PCR showed high expression in both the central brain and the optic lobes, consistent with the role of dopamine as neurotransmitter. Activation of TricaDop3 expressed in mammalian cells increased intracellular Ca²⁺ signalling and decreased NKH-477 (a forskolin analogue)-stimulated cyclic AMP levels in a dose-dependent manner. We studied the pharmacological profile of the TricaDop3 receptor and demonstrated that the synthetic vertebrate dopamine receptor agonists, 2 – amino- 6,7 – dihydroxy – 1,2,3,4 – tetrahydronaphthalene hydrobromide (6,7-ADTN) and bromocriptine acted as agonists. Methysergide was the most potent of the antagonists tested and showed competitive inhibition in the presence of dopamine. This study offers important information on the Dop3 receptor from Tribolium castaneum that will facilitate functional analyses of dopamine receptors in insects and other invertebrates.     

A new G protein-coupled receptor from a primitive metazoan shows homology with vertebrate aminergic receptors and displays constitutive activity in mammalian cells

Biogenic amine receptors mediate wide-ranging hormonal and modulatory functions in vertebrates, but are largely unknown in primitive invertebrates. In a representative of the most basal multicellular animals possessing a nervous system, the cnidarian Renilla koellikeri, aminergic-like receptors were previously characterized pharmacologically and found to engender control of the animal's bioluminescent and peristaltic reactions. Using degenerate oligonucleotides in a RT-PCR strategy, we obtained a full-length cDNA encoding a polypeptide with typical G protein-coupled receptor (GPCR) characteristics and which displayed a significant degree of sequence similarity (up to 45%) to biogenic amine receptors, particularly dopamine and adrenergic receptors. The new receptor, named Ren1, did not resemble any one specific type of amine GPCR and thus could not be identified on the basis of sequence. Ren1 was expressed transiently and stably in cultured mammalian cells, as demonstrated by immunocytochemistry and western blotting. Functional analysis of transfected HEK293, LTK- and COS-7 cells, based on both cAMP and Ca2+ signalling assays, revealed that Ren1 was not activated by any of the known biogenic amines tested and several related metabolites. The results indicated, however, that cells stably expressing Ren1 contained, on average, an 11-fold higher level of cAMP than the controls, in the absence of agonist stimulation. The high basal cAMP levels were shown to be specific for Ren1 and to vary proportionally with the level of Ren1 expressed in the transfected cells. Taken together, the data suggested that Ren1 was expressed as a constitutively active receptor. Its identification provides a basis for examination of the early evolutionary emergence of GPCRs and their functional properties.