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

Summary 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 “α-adrenergic-like octopamine receptors (OctαRs)”, “β-adrenergic-like octopamine receptors (OctβRs)” 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.     

A possible new class of octopamine receptors coupled to adenylate cyclase in the brain of the dipterous Ceratitis capitata. Pharmacological characterization and regulation of 3H-octopamine binding

Octopamine exerts its effects in insects through interaction with at least two classes of receptors, designated octopamine-1 and octopamine-2. Octopamine-2 receptors are positively coupled to adenylate cyclase, while octopamine-1 receptors are not coupled to this enzyme system. Ceratitis capitata brain appears to have octopamine receptors as unique aminergic receptors coupled to adenylate cyclase. These receptors show some pharmacological analogies with respect to octopamine-2 receptors, however they should constitute a new class of octopamine receptors. C. capitata brain octopamine receptors have also been characterized by [3H]octopamine-binding studies, exhibiting similar regulatory mechanisms to other receptors coupled to adenylate cyclase activation.     

Tyramine injections reduce locust viability

In the locust nervous system, tyramine is the direct precursor for octopamine synthesis and, as an octopamine analogue, it can activate octopamine receptors. Furthermore, the identification of specific tyramine receptors in Locusta migratoria and Drosophila melanogaster suggests that it is an important transmitter or modulator candidate. In this paper, we report that repeated tyramine injections reduced the viability of last instar larvae of Locusta and Schistocerca. In addition, a retardation of the last ecdysis was observed as a sublethal effect of the repeated tyramine treatment. Moreover, egg deposition by adult females was also retarded and/or drastically reduced. These effects show similarity to sublethal effects described for certain "insecticidal" octopamine receptor agonists, such as formamidines and phenyliminoimidazolidines. Since certain formamidine compounds were also shown to be agonists for the cloned tyramine receptors, it cannot be excluded that some lethal or sublethal consequences of tyramine administration are the result of an interaction with specific tyramine receptors.     

Identification and characterization of a novel family of Drosophila beta-adrenergic-like octopamine G-protein coupled receptors

Insect octopamine receptors carry out many functional roles traditionally associated with vertebrate adrenergic receptors. These include control of carbohydrate metabolism, modulation of muscular tension, modulation of sensory inputs and modulation of memory and learning. The activation of octopamine receptors mediating many of these actions leads to increases in the levels of cyclic AMP. However, to date none of the insect octopamine receptors that have been cloned have been convincingly shown to be capable of directly mediating selective and significant increases in cyclic AMP levels. Here we report on the identification and characterization of a novel, neuronally expressed family of three Drosophila G-protein coupled receptors that are selectively coupled to increases in intracellular cyclic AMP levels by octopamine. This group of receptors, DmOct beta1R (CG6919), DmOct beta2R (CG6989) and DmOct beta3R (CG7078) shows homology to vertebrate beta-adrenergic receptors. When expressed in Chinese hamster ovary cells all three receptors show a strong preference for octopamine over tyramine for the accumulation of cyclic AMP but show unique pharmacological profiles when tested with a range of synthetic agonists and antagonists. Thus, the pharmacological profile of individual insect tissue responses to octopamine might vary with the combination and the degree of expression of the individual octopamine receptors present.     

A new octopamine receptor class in locust nervous tissue, the octopamine 3 (OA3) receptor.

The insect neuronal 3H-octopamine binding site represents a new type of octopamine receptor. This receptor has pharmacological features that are characteristic for all known octopamine receptors, but it is possible to distinguish this receptor class from all others using either agonists or antagonists. The quantitative determination of the pharmacological relationships to the other octopamine receptor classes could demonstrate greatest homology with both class 2 (OA2A and OA2B) receptors. Therefore, the neuronal octopamine receptor should be named a class 3 receptor (OA3). A new and simple classification scheme for octopamine receptors which enables classification of the new receptor class is established using antagonists.     

Molecular and functional characterization of an octopamine receptor from honeybee (Apis mellifera) brain

Biogenic amines and their receptors regulate and modulate many physiological and behavioural processes in animals. In vertebrates, octopamine is only found in trace amounts and its function as a true neurotransmitter is unclear. In protostomes, however, octopamine can act as neurotransmitter, neuromodulator and neurohormone. In the honeybee, octopamine acts as a neuromodulator and is involved in learning and memory formation. The identification of potential octopamine receptors is decisive for an understanding of the cellular pathways involved in mediating the effects of octopamine. Here we report the cloning and functional characterization of the first octopamine receptor from the honeybee, Apis mellifera. The gene was isolated from a brain-specific cDNA library. It encodes a protein most closely related to octopamine receptors from Drosophila melanogaster and Lymnea stagnalis. Signalling properties of the cloned receptor were studied in transiently transfected human embryonic kidney (HEK) 293 cells. Nanomolar to micromolar concentrations of octopamine induced oscillatory increases in the intracellular Ca2+ concentration. In contrast to octopamine, tyramine only elicited Ca2+ responses at micromolar concentrations. The gene is abundantly expressed in many somata of the honeybee brain, suggesting that this octopamine receptor is involved in the processing of sensory inputs, antennal motor outputs and higher-order brain functions.     

Characterization of insect neuronal octopamine receptors (OA3 receptors)

Octopamine receptors in the nervous tissue of insects were investigated using a ligand-receptor assay with [³H]NC-5Z or [³H]octopamine as the radioligands. Both ligands recognized a homogenous class of binding sites with the properties of an octopamine receptor. This receptor has been characterized pharmacologically. Both high-affinity agonists (e.g. NC 7, K₁=0.3 nM) and antagonists (e.g. maroxepine, K₁=1.02 nM) were investigated. The neuronal octopamine receptor belongs to a receptor class that can easily be distinguished from peripheral octopamine receptors. Initial investigations of the localization of octopamine receptors within the insect nervous tissue show the greatest receptor density in the optic lobes.     

Evidence for octopaminergic modulation of an insect visceral muscle

Two dorsal unpaired median neurons (DUMOV1 and DUMOV2) lying in the posterior region of the VIIth abdominal ganglion of Locusta migratoria have axons which project to the muscles of the oviducts. This study reports the presence of octopamine within isolated DUMOV cell bodies, as well as in the oviducal nerve and innervated oviducal muscle. Individual cell bodies were pooled and found to contain about 0.34 pmol of octopamine per cell body giving an approximate value of 1.27 mM octopamine. Octopamine is concentrated within the area of oviducal muscle which receives DUMOV axons. Pharmacological studies reveal that the amplitude of neurally-evoked contractions of the oviducal muscle is reduced in a dose-dependent manner by octopamine, with threshold lying between 5 X 10(-10) M and 7 X 10(-9) M. The receptors for this response show a specificity for octopamine and synephrine, with an order of potency being octopamine = synephrine greater than metanephrine greater than tyramine greater than dopamine. The presence of octopamine throughout this neural pathway, coupled with the demonstration of octopaminergic modulation of muscular contraction, supports the hypothesis that octopamine serves a physiological role in this visceral system.     

Agonist-induced desensitization of an octopamine receptor

The octopamine-sensitive adenylate cyclase associated with haemocytes of the American cockroach, Periplaneta americana, has been used as a model system with which to study desensitization of the octopamine receptor. Preincubation of the haemocytes with octopamine results in a large decrease in subsequent maximal stimulation of cyclic AMP production by octopamine with little change in affinity of the receptor for the agonist. This effect of preincubation is dependent upon the concentration of octopamine in the preincubation media and on the duration of exposure. The attenuation appears to be a receptor-mediated event rather than an artifact of the preincubation. Octopamine receptor agonists (octopamine, synephrine, N-demethylchlordimeform) induce desensitization while biogenic amines with poor octopamine receptor affinity (dopamine, serotonin, norepinephrine) are without affect. In contrast, the octopamine receptor antagonist, phentolamine, appears to enhance subsequent stimulation by octopamine. The attenuation of octopamine stimulation of adenylate cyclase is conserved in broken-cell preparations with no alteration of responses to NaF or forskolin. Incubation of the cells with dibutyryl cyclic AMP or forskolin does not induce desensitization. The data indicate that the OA receptors coupled to AC in cockroach haemocytes undergo an homologous desensitization in response to exposure to agonists.     

Presynaptic modulation by octopamine at a single neuromuscular junction in the mealworm (Tenebrio molitor)

The effect of octopamine on the neuromuscular junction of the mealworm (Tenebrio molitor) was examined. Octopamine potentiated excitatory junctional potentials (EJPs) recorded intracellularly and extracellularly from ventral longitudinal muscle fibers. The potentiating action of octopamine was blocked in the presence of the alpha-adrenergic blocking agent, phentolamine, but not in the presence of another alpha-blocker, phenoxybenzamine, or the beta-blockers propranolol and dichloroisoproterenol. The resting membrane potential, membrane input resistance, reversal potential of EJP, glutamate potentials, and spontaneous miniature EJPs were found to be unaffected by octopamine. In contrast, quantal contents estimated by the extracellularly recorded EJP failures were greatly increased by octopamine. These results suggest that octopamine acted on the presynaptic terminals via alpha-adrenoceptor-like receptors (octopamine receptors) at the Tenebrio neuromuscular junctions to enhance the transmitter release associated with the motor nerve impulses.     

Octopamine- and dopamine-sensitive adenylate cyclase in the brain ofLocusta migratoria during its development

Octopamine- and dopamine-sensitive adenylate cyclases were studied in the brain of Locusta migratoria during its metamorphosis. In the adult brain the effects of octopamine and dopamine on adenylate cyclase were additive, suggesting the presence of separate populations of adenylate cyclase-linked receptors for octopamine and dopamine. There are no separate receptors for noradrenaline. Octopamine stimulates adenylate cyclase in both adult and larval brain; however, in adult brain octopamine is more potent than in larval brain. Dopamine stimulates adenylate cyclase activity only in adult brain. The sensitivity of adenylate cyclase to octopamine changes during the development of the animal. Phentolamine and cyproheptadine are potent antagonists of octopamine-stimulated adenylate cyclase, while propranolol has a weak effect. No cytosol factor which would modulate either basal or octopamine-stimulated adenylate cyclase was found. The effect of GTP and octopamine on adenylate cyclase was synergistic in adult brain but not in larval brain, while the effect of GppNHp and octopamine was synergistic in both adult and larval brains.     

A comparison of the signalling properties of two tyramine receptors from Drosophila

In invertebrates, the phenolamines, tyramine and octopamine, mediate many functional roles usually associated with the catecholamines, noradrenaline and adrenaline, in vertebrates. The α‐ and β‐adrenergic classes of insect octopamine receptor are better activated by octopamine than tyramine. Similarly, the Tyramine 1 subgroup of receptors (or Octopamine/Tyramine receptors) are better activated by tyramine than octopamine. However, recently, a new Tyramine 2 subgroup of receptors was identified, which appears to be activated highly preferentially by tyramine. We examined immunocytochemically the ability of CG7431, the founding member of this subgroup from Drosophila melanogaster, to be internalized in transfected Chinese hamster ovary (CHO) cells by different agonists. It was only internalized after activation by tyramine. Conversely, the structurally related receptor, CG16766, was internalized by a number of biogenic amines, including octopamine, dopamine, noradrenaline, adrenaline, which also were able to elevate cyclic AMP levels. Studies with synthetic agonists and antagonists confirm that CG16766 has a different pharmacological profile to that of CG7431. Species orthologues of CG16766 were only found in Drosophila species, whereas orthologues of CG7431 could be identified in the genomes of a number of insect species. We propose that CG16766 represents a new group of tyramine receptors, which we have designated the Tyramine 3 receptors.     

Characterisation of AmphiAmR4, an amphioxus (Branchiostoma floridae) α2-adrenergic-like G-protein-coupled receptor

Little is known about the evolutionary relationship between vertebrate adrenergic receptors and invertebrate octopamine and tyramine receptors. The complexity of the adrenergic signalling system is believed to be an innovation of the vertebrate lineage but the presence of noradrenaline has been reported in some invertebrate species. The cephalochordate, amphioxus (Branchiostoma floridae), is an ideal model organism for studying the evolution of vertebrate GPCRs, given its unique position at the base of the chordate lineage. Here, we describe the pharmacological characterisation and second messenger coupling abilities of AmphiAmR4, which clusters with α₂-adrenergic receptors in a phylogenetic tree but also shares a high sequence similarity to invertebrate octopamine/tyramine receptors in both BLAST and Hidden Markov Model analyses. Thus, it was of particular interest to determine if AmphiAmR4 displayed similar functional properties to the vertebrate α₂-adrenergic receptors or to invertebrate octopamine or tyramine receptors. When stably expressed in Chinese hamster ovary (CHO) cells, noradrenaline couples the receptor to both the activation of adenylyl cyclase and to the activation of the MAPKinase pathway. Pharmacological studies with a wide range of agonists and antagonists suggest that AmphiAmR4 functions as an α₂-adrenergic-like receptor when expressed in CHO cells.     

Octopamine receptor subtypes and their modes of action

Octopamine receptor subclasses were first proposed to explain differences in the pharmacological profiles of a range of physiological responses to octopamine obtained in the extensor-tibiae neuromuscular preparation of the locust. Thus, OCTOPAMINE₁ receptors which inhibit an endogenous myogenic rhythm, increase intracellular calcium levels. Also OCTOPAMINE₂ receptors which modulate neuromuscular transmission in this preparation, increase the level of adenylate cyclase activity. The current status of this classification is reviewed by examining the pharmacology of responses to octopamine in a range of preparations. It is concluded that the distinction between OCTOPAMINE₁ and OCTOPAMINE₂ receptor types is still valid, but that OCTOPAMINE₂ receptors exhibit some tissue specific variations. Studies on a clonedDrosophila octopamine/tyramine (phenolamine) receptor are discussed and illustrate many of the difficulties presently encountered in making a definitive classification of octopamine receptors. These include the possibilities that single receptors may activate multiple second messenger systems and that different agonists may differentially couple the same receptor to different second messenger systems.     

Characterization of a Prawn OA/TA Receptor in Xenopus Oocytes Suggests Functional Selectivity between Octopamine and Tyramine

Here we report the characterization of an octopamine/tyramine (OA/TA or TyrR1) receptor (OA/TA_Mac) cloned from the freshwater prawn, Macrobrachium rosenbergii, an animal used in the study of agonistic social behavior. The invertebrate OA/TA receptors are seven trans-membrane domain G-protein coupled receptors that are related to vertebrate adrenergic receptors. Behavioral studies in arthropods indicate that octopaminergic signaling systems modulate fight or flight behaviors with octopamine and/or tyramine functioning in a similar way to the adrenalins in vertebrate systems. Despite the importance of octopamine signaling in behavioral studies of decapod crustaceans there are no functional data available for any of their octopamine or tyramine receptors. We expressed OA/TA_Mac in Xenopus oocytes where agonist-evoked trans-membrane currents were used as readouts of receptor activity. The currents were most effectively evoked by tyramine but were also evoked by octopamine and dopamine. They were effectively blocked by yohimbine. The electrophysiological approach we used enabled the continuous observation of complex dynamics over time. Using voltage steps, we were able to simultaneously resolve two types of endogenous currents that are affected over different time scales. At higher concentrations we observe that octopamine and tyramine can produce different and opposing effects on both of these currents, presumably through the activity of the single expressed receptor type. The pharmacological profile and apparent functional-selectivity are consistent with properties first observed in the OA/TA receptor from the insect Drosophila melanogaster. As the first functional data reported for any crustacean OA/TA receptor, these results suggest that functional-selectivity between tyramine and octopamine is a feature of this receptor type that may be conserved among arthropods.     

Tyramine and octopamine: antagonistic modulators of behavior and metabolism

The phenolamines tyramine and octopamine are decarboxylation products of the amino acid tyrosine. Although tyramine is the biological precursor of octopamine, both compounds are independent neurotransmitters, acting through various G-protein coupled receptors. Especially, octopamine modulates a plethora of behaviors, peripheral and sense organs. Both compounds are believed to be homologues of their vertebrate counterparts adrenaline and noradrenaline. They modulate behaviors and organs in a coordinated way, which allows the insects to respond to external stimuli with a fine tuned adequate response. As these two phenolamines are the only biogenic amines whose physiological significance is restricted to invertebrates, the attention of pharmacologists was focused on the corresponding receptors, which are still believed to represent promising targets for new insecticides. Recent progress made on all levels of octopamine/tyramine research enabled us to better understand the molecular events underlying the control of complex behaviors.     

Cloning, expression and functional analysis of an octopamine receptor from Periplaneta americana

Octopamine regulates multiple physiological functions in invertebrates. The biological effects of octopamine and the pharmacology of octopamine receptors have been extensively studied in the American cockroach, Periplaneta americana. This paper reports the cloning of the first octopamine receptor from Periplaneta americana. A cDNA encoding a putative 7 transmembrane receptor was isolated from the head of Periplaneta americana. The encoded protein contains 628 amino acids and has sequence similarity to other biogenic amine receptors. This protein was expressed in COS-7 cells for radioligand binding studies using the antagonist 3H-yohimbine. Competitive binding comparing biogenic amines that could potentially function as endogenous ligands demonstrated this receptor had the highest affinity for octopamine (Ki = 13.3 microM) followed by tyramine, dopamine, serotonin and histamine. Octopamine increased both cAMP levels (EC50 = 1.62 microM) and intracellular concentrations of calcium through the receptor expressed in HEK-293 cells. Tyramine increased levels of both of these second messengers but only at significantly higher concentrations than octopamine. The cAMP increase by octopamine was independent of the increase in calcium. Competitive binding with antagonists revealed this receptor is similar to Lym oa1 from Lymnaea stagnalis. The data indicate that this cDNA is the first octopamine receptor cloned from Periplaneta americana and therefore has been named Pa oa1.     

In vitro and in vivo effects of formamidines in locust (Locusta migratoria migratorioides)

In vivo and in vitro experiments were used to study the effects of formamidines in the locust, Locusta migratoria migratorioides. In vivo the lethal and the antifeeding effects, in vitro the inhibition of the binding of a selective 3H-ligand to the receptors of octopamine, tyramine, dopamine, serotonin and gamma-amino butiric acid were studied. We have demonstrated that demethylchlordimeform is specific agonist to octopamine receptor, having high affinity to octopamine receptor, a moderate affinitiy to tyramine receptor and a low affinity to dopamine, serotonin and to gamma-amino butiric acid receptors. The demethylated chlordimeform analogoues, demethylchlordimeform and didemethylchlordimeform have higher affinity to the octopamine receptor than the parent compound. The formamidines had a toxic and an antifeeding effects when injected into the locust. The half lethal doses (LD50) and the feeding inhibition were correlated with the affinity of the compounds (Ki). The ring substitutions of the mulecule have alterated the both affinity and in vivo effect of the compounds. The most effective ring substitution pattern is 2,4-disubstitution with a combination of methyl groups or halogens. Our results suggest that the lethal effect of formamidines is mediated through the octopamine receptor.     

Dopamine counteracts octopamine signalling in a neural circuit mediating food response in C. elegans

Animals assess food availability in their environment by sensory perception and respond to the absence of food by changing hormone and neurotransmitter signals. However, it is largely unknown how the absence of food is perceived at the level of functional neurocircuitry. In Caenorhabditis elegans, octopamine is released from the RIC neurons in the absence of food and activates the cyclic AMP response element binding protein in the cholinergic SIA neurons. In contrast, dopamine is released from dopaminergic neurons only in the presence of food. Here, we show that dopamine suppresses octopamine signalling through two D2‐like dopamine receptors and the G protein Gi/o. The D2‐like receptors work in both the octopaminergic neurons and the octopamine‐responding SIA neurons, suggesting that dopamine suppresses octopamine release as well as octopamine‐mediated downstream signalling. Our results show that C. elegans detects the absence of food by using a small neural circuit composed of three neuron types in which octopaminergic signalling is activated by the cessation of dopamine signalling.