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Keywords:

  • cyclic AMP;
  • Drosophila;
  • expression in Chinese hamster ovary cells;
  • GPCR ;
  • receptor internalization;
  • tyramine

Abstract

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

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.

Abbreviations used
CHO

Chinese hamster ovary

ERK

extracellular-signal-related kinase

GFP

green fluorescent protein

GPCR

G-protein-coupled receptor

MAPK

mitogen-activated protein kinase

ORF

open reading frame

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.

Materials and methods

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

Cell culture, transfection and creation of stable cell lines

V5-His-tagged versions of the CG7431 and CG16766 receptors were generated by cloning appropriate PCR products into the expression vector pcDNA3.1 using the pcDNA3.1/V5-His/TOPO TA expression kit (Invitrogen Life Sciences, Paisley, UK) using the approach described previously (Maqueira et al. 2005). CHO-K1 cells were grown in F-12 medium supplemented with 10% CS-foetal bovine serum (FBS) and antibiotics (400 μg/mL G418, 100 U/mL penicillin, 100 μg/mL streptomycin) at 37°C and 5% CO2. After transfection of CG7431 or CG16766 cDNA into the cells using Lipofectamine 2000 reagent (Invitrogen), the antibiotic G418 (1.0 mg/mL) was added to the medium to select for cells that stably expressed the receptors. After 2 weeks of G418 selection, 15–20 G418-resistant colonies were trypsinized in cloning cylinders and transferred to 12-well plastic plates for expansion. These individual cell lines were analysed for receptor expression by western blot and by immunofluorescence. Immunocytochemistry was performed with an anti-V5-FITC antibody (Invitrogen) to confirm the expression of CG7431 and CG16766 in the CHO-K1 cell lines and to identify the receptor's localization within the cells at different times after exposure to putative endogenous agonists. The clonal cell lines that most efficiently expressed CG7431 and CG16766 were chosen for this study.

Detection and visualization of v5-tagged CG7431 and CG16766 receptors

V5-tagged CG7431 and CG16766 receptors were detected using immunocytochemistry on cell monolayers. Trypsinized cells were plated onto glass cover slips in six-well dishes at a density of 2 × 105 cells per well in 2 mL 10% CS-FBS/F-12 plus antibiotics (400 μg/mL G418, 100 U/mL penicillin, 100 μg/mL streptomycin). After 2 days, cells were stimulated with agonists (tyramine, octopamine, phenylethylamine, dopamine, synephrine, histamine, noradrenaline, adrenaline, 5-hydroxytryptamine, proctolin, ecdysone and 20-hydroxyecdysone) at the concentration of 1 μM or 10 μM for various times (from 5 min up to 3 h) and fixed with freshly prepared 4% paraformaldehyde in phosphate-buffered saline (PBS) for 10 min at 23°C. Transfected cells were permeabilized with 0.1% Triton in PBS for 5 min at 23°C and to reduce the non-specific binding, washed and then incubated in blocking solution (PBS containing 10% FBS) for 20 min. Cells were subsequently incubated with an anti-v5 tag FITC-conjugated antibody (20 mg/mL) (Invitrogen) for 60 min at 23°C (1 : 500 in blocking solution). For the colocalization experiments, cells were incubated with the following primary rabbit antibodies; anti-clathrin, anti-rab7, anti-rab11 (all purchased from Cell Signaling Technology, Boston, MA, USA), anti-LAMP1 and anti-TGN46 (both from Abcam, Cambridge, UK), following the manufacturer's instructions. An alexa fluor 555 donkey anti-rabbit secondary antibody (Invitrogen) (incubated 1 : 500 in blocking solution for 60 min at, 23°C) was used for their detection. Coverslips were then washed (five times in PBS) and mounted with Vectashield Mounting Media with DAPI (4′, 6-diamidino-2-phenylindole) onto glass slides. Cells were examined under an oil-immersion objective using an Olympus FV1000 confocal laser microscope (Olympus UK Ltd., Southend-on-Sea, UK).

cAMP determination

cAMP levels in stably transfected CHO-K1 cells were determined as described previously (Maqueira et al. 2005; Burman et al. 2009; Burman and Evans 2010), except 100 μM IBMX was used. cAMP levels are represented as a percentage of basal samples unless otherwise stated. Values for each concentration tested were measured in duplicate three times from three different batches of cells. Unless otherwise stated, all data are shown as mean ± SEM. Significance between two sets of data were tested using an unpaired, two-tailed t-test. Forskolin was used both to increase basal cAMP levels to make it easier to detect increases and decreases in cAMP levels in the same experiments and also to potentiate responses to agonists to more accurately determine their threshold effects (see Insel and Ostrom, 2003). A non-saturating 10-μM concentration of forskolin was used. Basal levels of CHO cell cyclic AMP were 5.1 ± 1.7 pmoles/mg protein (n = 3) and these were raised to 1053.7 ± 4.8 pmoles/mg protein after exposure to 10 μM forskolin. Protein levels were determined using a Bradford assay (Bradford 1976). The dose–response curves were drawn and EC50 values were determined using GraphPad PRISM 5 software (GraphPad Software Inc., La Jolla, CA, USA) using a non-linear regression curve-fitting programme.

Intracellular calcium measurements

Stably transfected CHO cells were assayed for changes in intracellular calcium levels using an Olympus Cell^R imaging system and the fluorescent indicator Fura-2, as described by Walker et al. (2008). However, a 60x objective was used and the experiments were carried out at 23°C. The Fura-2 was excited at 340 nm and 380 nm wavelengths and images were captured every second for 5 min. At 1 min into the experiment, a buffer control was added, and at 2 min into the experiment, the specified concentration of agonist was added for 10 s. A single coverslip of cells was used per concentration of agonist, and recordings were taken from up to 20 cells per field of view. Cells were chosen at random when greater than 20 cells responded per field of view. The results from each coverslip gave an n = 1 and each agonist concentration was repeated on at least five different days. Experiments were calibrated using 10 μM ionomycin and 4 mM EGTA, as described by Bootman and Roderick (2011). One coverslip of cells per imaging day was used for the calibration.

Phylogenetic tree

The phylogenetic tree was drawn using the Phylogeny.fr package (http://www.phylogeny.fr/). The receptor sequences were aligned using MUSCLE and the phylogeny analysed with PhyML (max bootstrap = 500). The tree was drawn using TreeDyn and the branch support values are expressed as percentages. Further details can be found in Dereeper et al. (2008, 2010). The tree was rooted using the sequence of the Drosophila proctolin receptor (CG6986).

Drugs

The drugs used in these experiments were obtained from the following sources: dopamine hydrochloride, ecdysone, 20-Hydroxy-ecdysone, 17-β-estradiol, Leu-enkephalin, ()-noradrenaline hydrochloride, ()-adrenaline, tyramine hydrochloride, (±)-p-octopamine hydrochloride, (±)-synephrine, (±)-1-phenylethylamine, phenylethanolamine, proctolin, 5-HT, histamine dihydrochloride, IBMX (3-Isobutyl methyl xanthine), clonidine hydrochloride, chlorpromazine hydrochloride, cyproheptadine hydrochloride, halostachine [α-(methylaminomethyl) benzyl alcohol], (±)-isoproterenol hydrochloride, naphazoline hydrochloride, metoclopramide hydrochloride, mianserin hydrochloride, phentolamine hydrochloride, (R)-(−)-phenylephrine hydrochloride, prazosin hydrochloride, promethazine hydrochloride, DL-propranolol, tolazoline, L-tyrosine and yohimbine hydrochloride were from Sigma-Aldrich (Poole, Dorset, UK); Forskolin was obtained from Abcam Biochemicals and Fura 2-AM were purchased from Tocris Bioscience (Bristol, UK).

Results

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

Agonist-dependent internalization of receptors

We initially undertook a comparative internalization study of the Drosophila CG7431 and CG16766 putative tyramine receptors using stable expression of C-terminal V5-tagged versions of the receptors in CHO cells combined with fluorescent antibody detection of the cellular location of the receptors. We used this approach as we would be able to measure receptor activation by different biogenic amine-related agonists independent of the specific second messenger pathways activated by the receptor/agonist complexes.

Confocal laser scanning microscopy indicates that under basal conditions, the CG7431 receptor was expressed preferentially on the cell surface of the CHO cell lines stably expressing the receptor, with some labelling on intracellular membranes (Fig. 1a). However, exposure of the cells to 1 μM tyramine for 3 h lead to a loss of labelling in the plasma membrane and increased labelling in the intracellular membrane compartment (Fig. 1b). Exposure to 10 μM tyramine for the same time period lead to an intense localization of receptor in a perinuclear compartment in most cells (Fig. 1c). Internalization to the perinuclear compartment was observed after only a 1 h incubation with 10 μM tyramine in some cells, but was maximal between 2 and 3 h. Thus, in parallel experiments to investigate the amine specificity of this agonist-induced internalization, cells were exposed to related biogenic amine agonists at a concentration of 10 μM for a period of 3 h. Figure 1d–f indicate that under such conditions no internalization to the perinuclear compartment was observed after exposure to octopamine, phenylethylamine or tyrosine. In parallel experiments under the same conditions, internalization was also not observed after exposure to synephrine, dopamine, noradrenaline, adrenaline, ecdysone or 20-OH-ecdysone. Similarly, internalization was not observed under these conditions after exposure to the tyrosine-containing peptides proctolin or Leu-enkephalin. Thus, the CG7431 receptor appears to be specifically internalized after exposure to tyramine consistent with the observation that this receptor can be specifically activated to generate an intracellular calcium signal when expressed in CHO cells or in Xenopus oocytes (Cazzamali et al. 2005).

image

Figure 1. The CG7431 receptor is only internalized after exposure of the stably transfected Chinese hamster ovary (CHO)-K1 cells to tyramine. (a) Basal. Cells were exposed to amines for 3 h. Exposure to 1 μM tyramine (b) resulted in increased internal presence in the endoplasmic reticulum, while exposure to 10 μM tyramine (c) resulted in an accumulation of receptor in a discrete perinuclear region. Exposure to 10 μM octopamine (d), phenylethylamine (e) or tyrosine (f) did not result in any receptor internalization from the plasma membrane. Expression of the v5-tagged receptor is shown in green. The nucleus is counter-stained with DAPI (blue).

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Cazzamali et al. (2005) predicted, on the basis of sequence similarity, that another aminergic-like Drosophila GPCR, CG16766, was also likely to be a tyramine-activated receptor, but presented no experimental evidence to support this conclusion. Furthermore, Huang et al. (2009) in their studies on the species orthologue of CG7431 from Bombyx mori, BmTAR2, noted that CG16766 while showing some structural similarities to the Type 2 Tyramine receptors, did not group together with them in a phylogenetic tree. Thus, we carried out a parallel internalization study with a CHO cell line stably expressing a V5-tagged version of the CG16766 receptor. Under basal conditions, this receptor was again localized at both the plasma membrane and in internal membranes within the transfected cells (Fig. 2a). Similar to CG7431, the CG16766 receptor was also internalized to the perinuclear compartment after exposure of the cells to 10 μM tyramine for 3 h (Fig. 2b). However, in contrast to CG7431, CG16766 was also internalized to the perinuclear compartment after exposure to similar concentrations of other biogenic amines, including octopamine, dopamine, noradrenaline, adrenaline (see Fig. 2c–f) and also synephrine and phenylethylamine (data not shown).

image

Figure 2. The CG16766 receptor was rapidly internalized into a perinuclear localization after exposure to 10 μM amines for a period of 3 h (a = Basal; b = tyramine; c = dopamine; d = adrenaline; e = noradrenaline; and f = octopamine). Expression of the v5-tagged receptor is shown in green. The nucleus is counter-stained with DAPI (blue).

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The internalization induced into the perinuclear compartment by 10 μM tyramine was first observed in some cells between 5 and 20 min of incubation and reached a peak between 30 min to 1 h (see Figure S1). The internalization observed after 2 and 3 h of exposure to tyramine was similar to that seen after 1 h of exposure. To determine the nature of the perinuclear compartment where the internalized receptor was accumulating, we performed colocalization studies of the tagged receptor with a number of intracellular compartmental markers (Fig. 3). It can be seen that after 30-min exposure to 10 μM tyramine CG16766 strongly colocalizes with clathrin (Fig. 3a) suggesting that it is internalized by a clathrin-dependent pathway. After a similar exposure to tyramine, the CG16766 receptor also showed some colocalization with Rab7 (Fig. 3b), a late endosomal marker, and with LAMP1 (Fig. 3c), a lysosomal marker. The receptor was also colocalized in the perinuclear compartment with TGN46, a trans-golgi network marker (Fig. 3d). Under the same conditions, the CG16766 receptor did not colocalize with the Rab11 (Figure S2), an early endosomal marker. No colocalization with any of the above compartmental markers could be found in control cells that were not stimulated by exposure to tyramine (Figure S3).

image

Figure 3. Colocalization of v5-tagged CG16766 receptor (green) with a range of intracellular markers (red) after exposure to 10 μM tyramine for 30 min (a = clathrin; b = Rab7, a late endosomal marker; c = LAMP1, a lysosome marker; and d = TGN46, a marker of the trans-golgi network). The nucleus is counter-stained with DAPI (blue) in the merged images.

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Thus, it would appear, that in contrast to CG7431, CG16766 is likely to be activated by a much larger number of biogenic amines in a fashion more comparable with the so-called Type 1 Tyramine receptors. The latter group includes the Drosophila Octopamine/Tyramine receptor (CG7485) (Robb et al. 1994) and the Bombyx mori Type 1 Tyramine receptor, BmTAR1 (Ohata et al. 2003). To quantify the interaction of biogenic amines with the putative Drosophila tyramine receptor, CG16766, we next examined the ability of the activated receptor to alter a number of second messenger levels in the transfected cells in a number of functional assays.

Amine specificity of modulation of intracellular cyclic AMP and calcium levels

We next compared the abilities of related biogenic amines to alter intracellular cyclic AMP levels in CHO cell lines stably expressing either the CG7431 or CG16766 putative tyramine receptors. Figure 4a indicates that exposure of the CG7431 expressing CHO cell line to 1 μM concentrations of synephrine, octopamine, tyramine, phenylethylamine, adrenaline, noradrenaline, dopamine or histamine did not produce any increases or decreases in forskolin-stimulated intracellular cyclic AMP levels. In addition, none of the amines tested altered forskolin-stimulated cyclic AMP levels in non-transfected wild-type CHO cells (see Supplementary data Bayliss and Evans 2012). Similarly, intracellular cyclic AMP levels were not altered in these cells after exposure to ecdysone (104.3 ± 2.8% of basal level, n = 3), 20-OH ecdysone (104.4 ± 9.7% of basal levels, n = 3), 17-β estradiol (93.5 ± 5.3% of basal levels, n = 3), proctolin (99.8 ± 5.3% of basal levels, n = 3) or tyrosine (100.2 ± 13.2% of basal levels, n = 3).

image

Figure 4. Effect of biogenic amines on forskolin-stimulated cAMP levels in CG7431-expressing (a) and CG16766-expressing (b and c) CHO-K1 cells. Cells were pre-incubated with 100 μM IBMX for 20 min, followed by incubation with 1 μM concentrations of various biogenic amines (a and b) or increasing concentrations of active biogenic amines (c) agonists, 10 μM forskolin and 100 μM IBMX for a further 20 min. Data are expressed as the mean ± SEM. > 3. SYN, Synephrine; OCT, Octopamine; TYR, Tyramine; PEA, Phenylethylamine; ADR, Adrenaline; NA, Noradrenaline; DA, Dopamine; HIS, Histamine; HAL, Halostachine; and PEOA, Phenylethanolamine.

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In contrast, intracellular levels of cyclic AMP were reduced in CHO cells stably expressing CG16766 (see Fig. 4b) after exposure to 1 μM tyramine (reduced to 57.5 ± 2.0% of basal levels, n = 36) or phenylethylamine (reduced to 53.3 ± 1.9% of basal levels, n = 12). Smaller reductions in intracellular cyclic AMP levels were also observed in these cells after exposure to 1 μM octopamine (reduced to 75.9 ± 3.0% of basal levels, n = 30), dopamine (reduced to 75.9 ± 4.0% of basal levels, n = 6) and synephrine (reduced to 79.3 ± 3.2% of basal levels, n = 12). In addition, intracellular cyclic AMP levels were not altered in these cells after exposure to ecdysone (106.0 ± 3.3% of basal level, n = 3), 20-OH ecdysone (102.5 ± 3.0% of basal levels, n = 3) and 17-β estradiol (118.0 ± 1.9% of basal levels, n = 3).

Full dose–response curves (Fig. 4c) indicate that the CG16766 receptor is maximally activated by tyramine (EC50 = 15.3 nM) and phenylethylamine (EC50 = 32.0 nM) with threshold concentrations for both amines occurring between 0.1 and 1.0 nM for an effect on forskolin-stimulated cyclic AMP levels. Octopamine was less potent (EC50 = 0.499 μM) with a threshold occurring between 0.1 and 1.0 μM. Octopamine was also only a partial agonist of the effects produced by tyramine and phenylethylamine on forskolin-stimulated cyclic AMP levels.

Full dose–response curves (Fig. 5) also indicate that tyramine (EC50 = 12.1 nM) and phenylethylamine (EC50 = 17.1 nM) were also able to mediate increases in intracellular calcium levels in CHO cell lines transfected with CG16766. Tyramine had a threshold between 0.1 and 1.0 nM and phenylethylamine between 1.0 and 3.0 nM for this effect.

image

Figure 5. Dose-dependent increases in intracellular calcium in Chinese hamster ovary (CHO) cells transfected with the CG16766 receptor after exposure to different concentrations of biogenic amines (tyramine = blue; phenylethylamine = green; octopamine = red; and dopamine = black). The results are expressed as the peak calcium responses (nM).

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Dopamine was less potent (EC50 = 0.93 μM) and octopamine was the least potent of the amines tested (EC50 = 4.9 μM). Both of the latter amines had thresholds between 100 and 300 nM. Interestingly, while octopamine was only a partial agonist of the effects of tyramine on the reduction of forskolin-stimulated cyclic AMP levels, it appeared to be a full agonist of the tyramine-mediated increase in peak calcium levels. Conversely, phenylethylamine appeared to be a full agonist of the effects on cyclic AMP levels while it was only a partial agonist of the peak calcium responses. A typical example of an individual calcium response to 10 μM tyramine is shown in Figure S4a, and a similar rank order of potency was obtained for the amines tested when measured as the percentage of cells responding with calcium increases in the field of view (Figure S4b). No increases in intracellular calcium were observed in non-transfected CHO cells with any of the amines tested up to a concentration of 10 μM.

Action of synthetic agonists and antagonists on modulation of intracellular cyclic AMP levels via CG16766

To further explore the pharmacological properties of CG16766 when expressed in CHO-K1 cells, we tested the ability of synthetic agonists to mimic the responses of the receptors to tyramine application (Fig. 6a) and the effects of synthetic antagonists to block the actions of tyramine on this receptor (Fig. 6c). We also constructed full dose–response curves for the agonists, tolazoline and naphazoline (Fig. 6b), which were full agonists of the tyramine-mediated changes in forskolin-stimulated cyclic AMP production. Their rank order of potency was Tolazoline (EC50 = 27.5 nM) > Naphazoline (EC50 = 60.96 nM). The α-adrenergic agonists, clonidine and phenylephrine, and the β-adrenergic agonist, isoproterenol, did not produce any changes in forskolin-stimulated cyclic AMP levels at concentrations up to 1 μM (Fig. 6a). None of the synthetic antagonists tested at a concentration of 1 μM was able to block the effects of 1 μM tyramine on forskolin-stimulated cyclic AMP levels. The β-adrenergic antagonist, propranolol, alone appeared to mimic the effects of tyramine-induced inhibition of adenylyl cyclase activity in the CG16766-transfected CHO-K1 cells. However, this effect was not likely to be CG16766 specific, as propranolol alone can also decrease forskolin-stimulated cAMP levels in wild-type CHO-K1 cells (Bayliss and Evans 2012).

image

Figure 6. Effects of synthetic agonists and tyramine (TYR) (a and b) on forskolin-stimulated cAMP levels in CG16766-expressing Chinese hamster ovary (CHO)-K1 cells. Cells were pre-incubated with 100 μM IBMX for 20 min, followed by incubation with 1 μM concentrations of agonists (a) or increasing concentrations of active agonists (b), 10 μM forskolin and 100 μM IBMX for a further 20 min. (c) CG16766-expressing CHO-K1 cells were pre-incubated with 100 μM IBMX for 20 min, followed by incubation with 1 μM tyramine, 10 μM forskolin (F) and 100 μM IBMX (open bars); 1 μM tyramine, 1 μM antagonist, 10 μM forskolin and 100 μM IBMX (hatched bars); or 1 μM antagonist, 10 μM forskolin and 100 μM IBMX (black bars) for a further 20 min. The basal value in the absence of agonist and antagonist is shown as 100%. Data are expressed as the mean ± SEM. > 3. NAPH, Naphazoline; TOLAZ, Tolazoline; CLON, Clonidine; PHEN, Phenylephrine; ISO, Isoprenaline; CHLOR, Chlorpromazine; CYPRO, Cyproheptadine; METO, Metoclopromide; PROM, Promethazine; PROP, Propranolol; YOH, Yohimbine; PHENT, Phentolamine; and MIAN, Mianserin.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. 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.

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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).

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

This work was supported by the BBSRC through the Babraham Institute and by a BBSRC Studentship to AB. GR was supported by a Leonardo Da Vinci Fellowship from the University of Rome and by a grant from the Babraham Institute. The authors declare no conflicts of interest.

References

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
  8. Supporting Information
FilenameFormatSizeDescription
jnc12158-sup-0001-FigS1-S4.pdfapplication/PDF626K

Figure S1. Time course of the v5-tagged CG16766 receptor internalization into a perinuclear location after exposure for different times to 10 μM tyramine.

Figure S2. Lack of co-localization of v5-tagged CG16766 receptor (green) and Rab 11 (red), an early endosome marker, under basal conditions (a) and after 30 min exposure to 10 μM tyramine (b).

Figure S3. Very little co-localization could be detected between the v5-tagged CG16766 receptor (green) and a range of intracellular markers (red) under basal conditions when the receptor was not activated.

Figure S4. CG16766 mediated changes in intracellular calcium mediated by exposure of receptor transfected CHO cells to biogenic amines.

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