Molecular cloning and expression analysis of the tyramine receptor genes in Apis cerana cerana.

Tyramine is a biological polyamine, which serves important functions as neurotransmitters, neuromodulators and neurohormone of the central nervous system. It participates in the regulation of various behavior and physiological processes in insects. For example, tyramine and its receptor genes are involved in the regulation of learning and memory in the animals. In this study, the full-length cDNA sequences of the tyramine receptor genes (Actyr1 and Actyr2) of the Chinese honeybee, Apis cerana cerana, were cloned and sequenced for the first time. Their expression patterns were examined in different tissues by qRT-PCR and localized in the head by in situ hybridization with digoxigenin (DIG)-labeled RNA probes. The full-length cDNAs of Actyr1 and Actyr2 are 1241 bp (GenBank accession no. KC814693) and 1270 bp (GenBank accession no.KC814693) in length and encode 297 amino acids and 399 amino acids, respectively. qRT-PCR results showed that the expression levels of both Actyr1 and Actyr2 were the highest in the head, followed by the abdomen, then the antennae and the lowest in the thorax. The expression level in the head was significantly higher than that in other tissues. Moreover, in situ hybridization showed that the expression of Actyr1 and Actyr2 genes were mainly localized to the Kenyon cells of the mushroom bodies and cells around the antennal lobes. These observations suggest that some interactions between these two genes in certain cells could be important in regulating various biological functions, such as learning and memory, in the honeybee.

Characterization of a ß-Adrenergic-Like Octopamine Receptor in the Oriental Fruit Fly, Bactrocera dorsalis (Hendel).

The biogenic amine octopamine plays a critical role in the regulation of many physiological processes in insects. Octopamine transmits its action through a set of specific G-protein coupled receptors (GPCRs), namely octopamine receptors. Here, we report on a ß-adrenergic-like octopamine receptor gene (BdOctßR1) from the oriental fruit fly, Bactrocera dorsalis (Hendel), a destructive agricultural pest that occurs in North America and the Asia-Pacific region. As indicated by RT-qPCR, BdOctßR1 was highly expressed in the central nervous system (CNS) and Malpighian tubules (MT) in the adult flies, suggesting it may undertake important roles in neural signaling in the CNS as well as physiological functions in the MT of this fly. Furthermore, its ligand specificities were tested in a heterologous expression system where BdOctßR1 was expressed in HEK-293 cells. Based on cyclic AMP response assays, we found that BdOctßR1 could be activated by octopamine in a concentration-dependent manner, confirming that this receptor was functional, while tyramine and dopamine had much less potency than octopamine. Naphazoline possessed the highest agonistic activity among the tested agonists. In antagonistic assays, mianserin had the strongest activity and was followed by phentolamine and chlorpromazine. Furthermore, when the flies were kept under starvation, there was a corresponding increase in the transcript level of BdOctßR1, while high or low temperature stress could not induce significant expression changes. The above results suggest that BdOctßR1 may be involved in the regulation of feeding processes in Bactrocera dorsalis and may provide new potential insecticide leads targeting octopamine receptors.

Octopamine and tyramine respectively regulate attractive and repulsive behavior in locust phase changes.

Aggregative and solitary behaviors are universal phenomena in animals. Interestingly, locusts (Locusta migratoria) can reversibly transit their behavior between gregarious and solitary phase through conspecific attraction and repulsion. However, the regulatory mechanism of neurotransmitters underlying attraction and repulsion among locusts remains unknown. In this study, we found gregarious and solitary locusts were attracted or repulsed respectively by gregarious volatiles. Solitary locusts can transform their preference for gregarious volatiles during crowding, whereas gregarious locusts avoided their volatiles during isolation. During crowding and isolation, the activities of octopamine and tyramine signalings were respectively correlated with attraction- and repulsion-response to gregarious volatiles. RNA interference verified that octopamine receptor α (OARα) signaling in gregarious locusts controlled attraction-response, whereas in solitary ones, tyramine receptor (TAR) signaling mediated repulsion-response. Moreover, the activation of OARα signaling in solitary locusts caused the behavioral shift from repulsion to attraction. Enhancement of TAR signaling in gregarious locusts resulted in the behavioral shift from attraction to repulsion. The olfactory preference of gregarious and solitary locusts co-injected by these two monoamines displayed the same tendency as the olfactory perception in crowding and isolation, respectively. Thus, the invertebrate-specific octopamine-OARα and tyramine-TAR signalings respectively mediate attractive and repulsive behavior in behavioral plasticity in locusts.

Honey bee dopamine and octopamine receptors linked to intracellular calcium signaling have a close phylogenetic and pharmacological relationship.

BACKGROUND: Three dopamine receptor genes have been identified that are highly conserved among arthropod species. One of these genes, referred to in honey bees as Amdop2, shows a close phylogenetic relationship to the a-adrenergic-like octopamine receptor family. In this study we examined in parallel the functional and pharmacological properties of AmDOP2 and the honey bee octopamine receptor, AmOA1. For comparison, pharmacological properties of the honey bee dopamine receptors AmDOP1 and AmDOP3, and the tyramine receptor AmTYR1, were also examined. METHODOLOGY/PRINCIPAL FINDINGS: Using HEK293 cells heterologously expressing honey bee biogenic amine receptors, we found that activation of AmDOP2 receptors, like AmOA1 receptors, initiates a rapid increase in intracellular calcium levels. We found no evidence of calcium signaling via AmDOP1, AmDOP3 or AmTYR1 receptors. AmDOP2- and AmOA1-mediated increases in intracellular calcium were inhibited by 10 µM edelfosine indicating a requirement for phospholipase C-ß activity in this signaling pathway. Edelfosine treatment had no effect on AmDOP2- or AmOA1-mediated increases in intracellular cAMP. The synthetic compounds mianserin and epinastine, like cis-(Z)-flupentixol and spiperone, were found to have significant antagonist activity on AmDOP2 receptors. All 4 compounds were effective antagonists also on AmOA1 receptors. Analysis of putative ligand binding sites offers a possible explanation for why epinastine acts as an antagonist at AmDOP2 receptors, but fails to block responses mediated via AmDOP1. CONCLUSIONS/SIGNIFICANCE: Our results indicate that AmDOP2, like AmOA1, is coupled not only to cAMP, but also to calcium-signalling and moreover, that the two signalling pathways are independent upstream of phospholipase C-ß activity. The striking similarity between the pharmacological properties of these 2 receptors suggests an underlying conservation of structural properties related to receptor function. Taken together, these results strongly support phylogenetic analyses indicating that the AmDOP2 and AmOA1 receptor genes are immediate paralogs.

Cloning and expression pattern of a putative octopamine/tyramine receptor in antennae of the noctuid moth Mamestra brassicae.

In insects, biogenic amines have been shown to play an important role in olfactory plasticity. In a first attempt to decipher the underlying molecular mechanisms, we report the molecular cloning and precise expression pattern of a newly identified octopamine/tyramine-receptor-encoding gene in the antennae of the noctuid moth Mamestra brassicae (MbraOAR/TAR). A full-length cDNA has been obtained through homology cloning in combination with rapid amplification of cDNA ends/polymerase chain reaction; the deduced protein exhibits high identities with previously identified octopamine/tyramine receptors in other moths. In situ hybridization within the antennae has revealed that MbraOAR/TAR is expressed at the bases of both pheromone-sensitive and non-sensitive olfactory sensilla and in cells with a neurone-like shape. In accordance with previous physiological studies that have revealed a role of biogenic amines in the electrical activity of the receptor neurones, our results suggest that biogenic amines (either octopamine or tyramine) target olfactory receptor neurones to modulate olfactory coding as early as the antennal level.

A new family of insect tyramine receptors.

The Drosophila Genome Project database contains a gene, CG7431, annotated to be an "unclassifiable biogenic amine receptor." We have cloned this gene and expressed it in Chinese hamster ovary cells. After testing various ligands for G protein-coupled receptors, we found that the receptor was specifically activated by tyramine (EC(50), 5x10(-7)M) and that it showed no cross-reactivity with beta-phenylethylamine, octopamine, dopa, dopamine, adrenaline, noradrenaline, tryptamine, serotonin, histamine, and a library of 20 Drosophila neuropeptides (all tested in concentrations up to 10(-5) or 10(-4)M). The receptor was also expressed in Xenopus oocytes, where it was, again, specifically activated by tyramine with an EC(50) of 3x10(-7)M. Northern blots showed that the receptor is already expressed in 8-hour-old embryos and that it continues to be expressed in all subsequent developmental stages. Adult flies express the receptor both in the head and body (thorax/abdomen) parts. In addition to the Drosophila tyramine receptor gene, CG7431, we found another closely related Drosophila gene, CG16766, that probably also codes for a tyramine receptor. Furthermore, we annotated similar tyramine-like receptor genes in the genomic databases from the malaria mosquito Anopheles gambiae and the honeybee Apis mellifera. These four tyramine or tyramine-like receptors constitute a new receptor family that is phylogenetically distinct from the previously identified insect octopamine/tyramine receptors. The Drosophila tyramine receptor is, to our knowledge, the first cloned insect G protein-coupled receptor that appears to be fully specific for tyramine.

Insect octopamine receptors: a new classification scheme based on studies of cloned Drosophila G-protein coupled receptors.

Insect octopamine receptors are G-protein coupled receptors. They can be coupled to second messenger pathways to mediate either increases or decreases in intracellular cyclic AMP levels or the generation of intracellular calcium signals. Insect octopamine receptors were originally classified on the basis of second messenger changes induced in a variety of intact tissue preparations. Such a classification system is problematic if more than one receptor subtype is present in the same tissue preparation. Recent progress on the cloning and characterization in heterologous cell systems of octopamine receptors from Drosophila and other insects is reviewed. A new classification system for insect octopamine receptors into "alpha-adrenergic-like octopamine receptors (OctalphaRs)", "beta-adrenergic-like octopamine receptors (OctbetaRs)" and "octopamine/tyramine (or tyraminergic) receptors" is proposed based on their similarities in structure and in signalling properties with vertebrate adrenergic receptors. In future studies on the molecular basis of octopamine signalling in individual tissues it will be essential to identify the relative expression levels of the different classes of octopamine receptor present. In addition, it will be essential to identify if co-expression of such receptors in the same cells results in the formation of oligomeric receptors with specific emergent pharmacological and signalling properties.

GPCRsclass: a web tool for the classification of amine type of G-protein-coupled receptors.

The receptors of amine subfamily are specifically major drug targets for therapy of nervous disorders and psychiatric diseases. The recognition of novel amine type of receptors and their cognate ligands is of paramount interest for pharmaceutical companies. In the past, Chou and co-workers have shown that different types of amine receptors are correlated with their amino acid composition and are predictable on its basis with considerable accuracy [Elrod and Chou (2002) Protein Eng., 15, 713-715]. This motivated us to develop a better method for the recognition of novel amine receptors and for their further classification. The method was developed on the basis of amino acid composition and dipeptide composition of proteins using support vector machine. The method was trained and tested on 167 proteins of amine subfamily of G-protein-coupled receptors (GPCRs). The method discriminated amine subfamily of GPCRs from globular proteins with Matthew's correlation coefficient of 0.98 and 0.99 using amino acid composition and dipeptide composition, respectively. In classifying different types of amine receptors using amino acid composition and dipeptide composition, the method achieved an accuracy of 89.8 and 96.4%, respectively. The performance of the method was evaluated using 5-fold cross-validation. The dipeptide composition based method predicted 67.6% of protein sequences with an accuracy of 100% with a reliability index > or =5. A web server GPCRsclass has been developed for predicting amine-binding receptors from its amino acid sequence [http://www.imtech.res.in/raghava/gpcrsclass/ and http://bioinformatics.uams.edu/raghava/gpersclass/ (mirror site)].

Dimerization in aminergic G-protein-coupled receptors: application of a hidden-site class model of evolution.

G-Protein-coupled receptors (GPCRs) are an important superfamily of transmembrane proteins involved in cellular communication. Recently, it has been shown that dimerization is a widely occurring phenomenon in the GPCR superfamily, with likely important physiological roles. Here we use a novel hidden-site class model of evolution as a sequence analysis tool to predict possible dimerization interfaces in GPCRs. This model aims to simulate the evolution of proteins at the amino acid level, allowing the analysis of their sequences in an explicitly evolutionary context. Applying this model to aminergic GPCR sequences, we first validate the general reasoning behind the model. We then use the model to perform a family specific analysis of GPCRs. Accounting for the family structure of these proteins, this approach detects different evolutionarily conserved and accessible patches on transmembrane (TM) helices 4-6 in different families. On the basis of these findings, we propose an experimentally testable dimerization mechanism, involving interactions among different combinations of these helices in different families of aminergic GPCRs.

Phylogenetic analysis of 277 human G-protein-coupled receptors as a tool for the prediction of orphan receptor ligands.

BACKGROUND: G-protein-coupled receptors (GPCRs) are the largest and most diverse family of transmembrane receptors. They respond to a wide range of stimuli, including small peptides, lipid analogs, amino-acid derivatives, and sensory stimuli such as light, taste and odor, and transmit signals to the interior of the cell through interaction with heterotrimeric G proteins. A large number of putative GPCRs have no identified natural ligand. We hypothesized that a more complete knowledge of the phylogenetic relationship of these orphan receptors to receptors with known ligands could facilitate ligand identification, as related receptors often have ligands with similar structural features. RESULTS: A database search excluding olfactory and gustatory receptors was used to compile a list of accession numbers and synonyms of 81 orphan and 196 human GPCRs with known ligands. Of these, 241 sequences belonging to the rhodopsin receptor-like family A were aligned and a tentative phylogenetic tree constructed by neighbor joining. This tree and local alignment tools were used to define 19 subgroups of family A small enough for more accurate maximum-likelihood analyses. The secretin receptor-like family B and metabotropic glutamate receptor-like family C were directly subjected to these methods. CONCLUSIONS: Our trees show the overall relationship of 277 GPCRs with emphasis on orphan receptors. Support values are given for each branch. This approach may prove valuable for identification of the natural ligands of orphan receptors as their relation to receptors with known ligands becomes more evident.