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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.
In invertebrates, the biogenic amines, octopamine and tyramine, carry out many of the functions usually associated with noradrenaline and adrenaline in vertebrates (Evans 1980; Roeder et al. 2003; Roeder 2005). Thus, in insects, octopamine has been shown to be involved in numerous physiological processes (Verlinden et al. 2010), including arousal and wakefulness (Bacon et al. 1995; Crocker and Seghal 2008), modulation of neuromuscular transmission (Evans and O'Shea 1977), modulation of the immune system (Adamo 2010), sensory modulation (Farooqui 2007), egg laying (Orchard and Lange 1985; Lee et al. 2003; Monastirioti 2003), associative learning and memory (Hammer and Menzel 1998; Schwaerzel et al. 2003) and the regulation of muscular energy metabolism (Mentel et al. 2003).
Tyramine, on the other hand, was initially considered as only a metabolic precursor for octopamine, which cross-reacted with receptors that were more specific for octopamine. However, evidence is now emerging for specific roles for tyramine in insects and nematodes, independent from those of octopamine. Thus, tyramine has been shown to cause opposite behavioural effects to octopamine in a number of insect preparations (Lange 2008). Furthermore, genetic studies have shown a role for tyramine in insect olfaction (Kutsukake et al. 2000) and on the inhibition of egg laying and the modulation of reversal behaviour and the suppression of head oscillations in response to touch in C. elegans (Alkema et al. 2005). The latter study also provided evidence for the existence of specific identified tyraminergic neurons mediating these effects. Immunocytochemical studies in locusts have also presented evidence for the existence of neurones expressing both octopamine and tyramine, which is not unexpected as tyramine is a metabolic precursor for octopamine (Kononenko et al. 2009). However, the same study also identified specific classes of neurones, which were immunoreactive to tyramine alone. Interestingly, some of the latter neurones could be induced to express octopamine when exposed to a variety of stressful conditions. Although the majority of the latter neurones did not respond to stress, and are thus likely to represent specific tyraminergic neurones, it cannot be ruled out that they may also express octopamine under specific behavioural and environmental conditions. However, to date in all the above studies, it is not clear if tyramine is released as the sole functional endogenous ligand from the proposed tyraminergic neurones (Alkema et al. 2005) or is coreleased with octopamine from the neurones that express both amines. Given the proposed opposite actions of octopamine and tyramine in some physiological processes, a behavioural modulation of the relative amounts of octopamine and tyramine released from a single neurone would be of much interest.
In the vast majority of cases, the actions of octopamine and tyramine have been shown to be mediated via the activation of G-protein-coupled receptors, but it should be noted that tyramine-gated chloride channels have been shown to mediate some of the above-mentioned actions of tyramine in C.elegans (Pirri et al. 2009). Invertebrate GPCRs, which bind octopamine and/or tyramine have recently been classified into three subtypes, α-adrenergic-like octopamine receptors (OctαRs), β-adrenergic-like octopamine receptors (OctβRs) and octopamine/tyramine (Oct/Tyr) or Tyramine 1 receptors (Evans and Maqueira 2005; Maqueira et al. 2005). The OctαRs and the OctβRs show a preference for octopamine, but are also activated by tyramine to a variable extent depending on the properties of the individual receptor and its species of origin. The Oct/Tyr receptors show a preference for tyramine over octopamine in binding studies and in the inhibition of adenylyl cyclase activity (hence the designation Tyramine 1 receptors). However, they have also been shown to exhibit agonist-specific coupling to other second messenger systems, such that the Drosophila version of this receptor is more sensitive to octopamine rather than tyramine in terms of calcium release from internal stores (see Robb et al. 1994). More recently, a second family of tyramine receptors (Tyramine 2 receptors) has been identified in Drosophila (CG7431) (Cazzamali et al. 2005), in Bombyx mori (BmTAR2) (Huang et al.2009) and bioinformatically in a number of other insect species. Characterization of this second group of tyramine receptors shows that they are highly specific for tyramine over octopamine, with octopamine only showing responses on BmTAR2 at concentrations above 100 μM. In functional terms, they have only been shown to be coupled to a release of calcium from internal stores. Cazzamali et al. (2005) also noted that the Drosophila genome also contained another putative tyramine receptor, CG16766, which was structurally related to CG7431, but CG16766 was not characterized in terms of its pharmacology and mode of action. The invertebrate tyramine receptors appear to have evolved separately from the vertebrate trace amine-associated receptors, which may also be activated by tyramine (Zucchi et al. 2006).
Here, we report on a comparative study of the pharmacology, internalization and second messenger coupling of the putative Drosophila tyramine receptors CG7431 and CG16766. We note that despite their strong sequence similarities, they have different pharmacologies, second messenger coupling capabilities and tissue expression patterns.
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- Materials and methods
- Supporting Information
The two putative Drosophila tyramine receptors, CG7431 and CG16766, lie adjacent to each other on the right arm of Chromosome 3 and have a very similar intron–exon structure with five coding exons. They share a 61% sequence identity and a 79% sequence similarity. They are much more similar to each other than to the Drosophila Oct/Tyr receptor (CG7485 or DmTAR1) (CG7431: 30% Identical and 47% Similar; CG16766: 44% Identical and 65% Similar). Thus, they are likely to be paralogue genes that have arisen by gene duplication. The results of this study suggest that the two genes are likely to have evolved different functions over the course of evolution.
CG7431 is a highly unusual insect biogenic amine receptor in that it shows a very high specificity for the biogenic amine tyramine in calcium-release assays when expressed in CHO cells (Cazzamali et al. 2005). Structurally related biogenic amines, such as octopamine, synephrine and dopamine did not show any effects up to a concentration of 100 μM on CG7431 (Cazzamali et al. 2005) or on its species homologue from Bombyx (BmTAR2) (Huang et al. 2009). We wondered if the Drosophila receptor might show agonist-specific coupling (Robb et al. 1994; Evans et al. 1995; Kenakin 1995, 2011) for different biogenic amines if it was assayed using different second messenger systems. However, neither tyramine nor any of the other agonists tested showed any effects in cyclic AMP assays up to a concentration of 1 μM. To attempt to determine if the receptor might be able to couple to some other untested second messenger pathway, we carried out a series of internalization studies to see if the receptor could be activated by biogenic amines other than tyramine. A considerable number of biogenic amine-activated GPCRs show agonist-dependent internalization (Shenoy and Lefkowitz 2003; Drake et al. 2006). However, in our studies, tyramine was the only biogenic amine to internalize the receptor from the plasma membrane to a perinuclear localization. As another Drosophila GPCR, CG 18314 or DmDopEcR, also shows a very high specificity for dopamine in cyclic AMP stimulation assays, but also responds to insect steroids such as ponasterone A, ecdysone and 20-hydroxy-ecdysone (Srivastava et al. 2005), we also explored the possibility that CG7431 might also be a steroid receptor. However, ecdysone, 20-hydroxy-ecdysone or 17β-estradiol were not able to induce the internalization of the receptor. Furthermore, as tyrosine-containing neuropeptides effectively contain a ‘tyramine motif” (Kuz'mina et al. 2006), we also tested the effectiveness of neuropeptides including, proctolin and Leu-enkephalin, for their ability to internalize CG7431. However, neither of the peptides was effective. Thus, CG7431 is either a highly unusual biogenic amine receptor with a very high specificity for tyramine or has a hitherto unknown endogenous agonist.
CG16766 showed a very different pharmacology to CG7431 in terms of both its agonist-mediated internalization and biogenic amine specificity. In both cases, several other biogenic amines were also effective, similar to the Drosophila Oct/Tyr receptor (Arakawa et al. 1990; Robb et al. 1994). Thus, tyramine and phenylethylamine were almost equally effective on the receptor, and their effects were reduced by adding a β-hydroxyl group and an N-terminal methyl group. Interestingly, phenylethylamine was also able to block the binding of [3H] tyramine to the BmTAR2 receptor, but was not able to activate it (Huang et al.2009). In terms of synthetic agonists, CG16766 showed some similarities with the locust Tyr 1 receptor (Vanden Broeck et al. 1995), the Bombyx Tyr 2 receptor (Huang et al. 2009) and the Drosophila β-adrenergic octopamine receptors (Maqueira et al. 2005) in that it was activated by the α-adrenergic agonists, naphazoline and tolazoline. In contrast to other insect octopamine and tyramine receptors, synthetic antagonists such as yohimbine, mianserin and chlorpromazine were not able to block the actions of tyramine on CG16766 (Evans and Robb 1993; Evans and Maqueira 2005).
CG7431 and CG16766 also differ in their reported tissue expression patterns in Drosophila (Chintapalli et al. 2007), further suggesting that they may undertake different physiological roles in the animal. The expression of CG7431 is enriched in the brain, thoracicoabdominal ganglion and in the midgut in the adult and in the CNS, tubule and hindgut in larvae. This suggests that it might have functional roles as a neurotransmitter or neuromodulatory receptor in the nervous system and in the control of gut function. The orthologous receptor BmTAR2 was detected in the brain and nerve cord of the fifth instar larvae, but not in the silk gland, midgut or Malpighian tubules (Huang et al. 2009). The expression of the CG16766 receptor, on the other hand, is highly enriched in the crop and eye in adults and in the tubule and hindgut in larvae. This suggests that it might have a specific role in the modulation of vision and in the storage of food. These data further suggest that the paralogous receptors may have gained different functions during the course of evolution.
A phylogenetic analysis of the Drosophila melanogaster CG7431 and CG16766 receptor sequences (Fig. 7) clearly shows that both receptor sequences group together with orthologous sequences from the other Drosophila species of the subgenus Sophophora, such as those of D. simulans, D. sechellia, D. yakuba and D erecta (Song et al. 2011). In addition, the CG7431 sequence also clusters with the Tyramine 2 receptors from a number of other insect species. However, we were not able to identify any homologous sequences to that of CG16766 in any of the sequenced insect genomes, confirming and extending the observation of Huang et al. (2009), who could not find a CG16766-like sequence in the Bombyx genome. Thus, CG16766 appears to be a sequence specific to the genus Drosophila and because of its different pharmacological properties to both Tyramine 1 and Tyramine 2 receptors, we propose that CG16766 be designated the founder member of a Tyramine 3 subgroup of receptors. Furthermore, the CG16766 sequence could potentially be used as a target site for the development of novel Drosophila-specific control agents.
Figure 7. Phylogenetic tree comparison of representative members of the different insect tyramine and octopamine receptor classes. The CG7431 receptor sequence clusters with the Tyramine 2 subgroup of receptors from other Drosophila species and other insect species. However, the CG16766 receptor sequence clusters with only the orthologous sequences from other Drosophila species in the proposed Tyramine 3 subgroup of receptors. The phylogenetic tree was constructed using the Phylogeny.fr package of programmes with maximum bootstrap = 500 and the branch support values expressed as a percentage (Dereeper et al. 2008, 2010). The tree was rooted with the Drosophila melanogaster proctolin (CG6986) sequence. Dm = Drosophila melanogaster; Dmoja, D.mojavensis; Dgrim, D.grimshawi; Dviril, D.virilis; Dpseud, D.pseudoobscura; Dpers, D.persimilis; Dana, D.ananassae; Dwill, D.willistoni; Dsim, D.simulans; Dsech, D.sechellia; Derecta, D.erecta; Dyak, D.yakuba; Tc, Tribolium castaneum; Am, Apis mellifera; Bm, Bombyx mori; Ag, Anopheles gambiae; Ap, Acyrthosiphon pisum; Hv, Heliothis virescens; Px, Papilio xuthus; Lm, Locusta migratoria; Rm, Rhipicephalus microplus; Ce, Caenorhabditis elegans.
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It would thus appear that in insects tyramine could bring about its physiological actions by potentially interacting with two or three groups of ‘tyramine receptors’ and/or with the two additional groups of ‘octopamine receptors’, namely the α-adrenergic- and β-adrenergic-like octopamine receptors (Evans and Maqueira 2005; Maqueira et al. 2005). To decide the exact physiological roles of tyramine in different locations in the insect nervous system, and in other tissues, it will be necessary to correlate the tissue-specific and stage-specific expression patterns of the above receptors with the release sites of both specific tyraminergic and octopaminergic neurons. Tyramine could potentially be released from both these types of neurones. Indeed, in a parallel situation in the CA1 field of the dorsal hippocampus, the release of dopamine from noradrenergic neurons has been suggested to be responsible for the activation of D1 dopamine receptors (Smith and Greene 2012).