Two splicing variants of a novel family of octopamine receptors with different signaling properties

Authors

  • Shun-Fan Wu,

    1. State Key Laboratory of Rice Biology & Key Laboratory of Agricultural Entomology of Ministry of Agriculture, Institute of Insect Sciences, Zhejiang University, Hangzhou, China
    2. Key Laboratory of Integrated Management of Crop Disease and Pests, Ministry of Education, Department of Pesticide Sciences, College of Plant Protection, Nanjing Agricultural University, Jiangsu Key Laboratory of Pesticide Sciences, Nanjing, China
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  • Gang Xu,

    1. State Key Laboratory of Rice Biology & Key Laboratory of Agricultural Entomology of Ministry of Agriculture, Institute of Insect Sciences, Zhejiang University, Hangzhou, China
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  • Yi-Xiang Qi,

    1. State Key Laboratory of Rice Biology & Key Laboratory of Agricultural Entomology of Ministry of Agriculture, Institute of Insect Sciences, Zhejiang University, Hangzhou, China
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  • Ren-Ying Xia,

    1. State Key Laboratory of Rice Biology & Key Laboratory of Agricultural Entomology of Ministry of Agriculture, Institute of Insect Sciences, Zhejiang University, Hangzhou, China
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  • Jia Huang,

    Corresponding author
    1. State Key Laboratory of Rice Biology & Key Laboratory of Agricultural Entomology of Ministry of Agriculture, Institute of Insect Sciences, Zhejiang University, Hangzhou, China
    • Address correspondence and reprint requests to Dr Jia Huang and Gong-Yin Ye, Institute of Insect Sciences, Zhejiang University, Yuhangtang Road 688, Hangzhou 310058, China. E-mails: huangj@zju.edu.cn and chu@zju.edu.cn

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  • Gong-Yin Ye

    Corresponding author
    1. State Key Laboratory of Rice Biology & Key Laboratory of Agricultural Entomology of Ministry of Agriculture, Institute of Insect Sciences, Zhejiang University, Hangzhou, China
    • Address correspondence and reprint requests to Dr Jia Huang and Gong-Yin Ye, Institute of Insect Sciences, Zhejiang University, Yuhangtang Road 688, Hangzhou 310058, China. E-mails: huangj@zju.edu.cn and chu@zju.edu.cn

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Abstract

The octopamine and tyramine, as the invertebrate counterparts of the vertebrate adrenergic transmitters, control and modulate many physiological and behavioral processes. Both molecules mediate their effects by binding to specific receptors belonging to the superfamily of G-protein-coupled receptors. So far, four families of octopamine and tyramine receptors have been reported. Here, we described the functional characterization of one putative octopamine/tyramine receptor gene from the rice stem borer, Chilo suppressalis. By a mechanism of alternative splicing, this receptor gene (CsOA3) encodes two molecularly distinct transcripts, CsOA3S and CsOA3L. CsOA3L differs from CsOA3S on account of the presence of an additional 30 amino acids within the third intracellular loop. When heterologously expressed, both receptors cause increases of intracellular Ca2+ concentration. The short form, CsOA3S, was activated by both octopamine and tyramine, resulting in decreased intracellular cAMP levels ([cAMP]i) in a dose-dependent manner, whereas dopamine and serotonin are not effective. However, CsOA3L did not show any impact on [cAMP]i. Studies with series of agonists and antagonists confirmed that CsOA3 has a different pharmacological profile from that of other octopamine receptor families. The CsOA3 is, to our knowledge, a novel family of insect octopamine receptors.

image

Octopamine, the invertebrate counterpart of noradrenaline, modulates many physiological processes. Four families of octopamine/tyramine receptors have been reported. We found that a novel family of octopamine receptors, which encodes two transcripts by alternative splicing, couple with different second messenger pathways. It implicated that one octopamine receptor gene could play different functional roles by alternative splicing.

Abbreviations used
CHO

Chinese hamster ovary

Cs

Chilo suppressalis

DMEM

Dulbecco's modified Eagle's medium

D-PBS

Dulbecco's phosphate buffered saline

FBS

fetal bovine serum

G418

gentamycin sulfate

GPCR

G-protein-coupled receptor

HEK293

human embryo kidney 293

IBMX

3-isobutyl-1-methyl xanthine

OA3 receptor

octopamine 3 receptor

OA

octopamine

ORF

open reading frame

TA

tyramine

TM

transmembrane

The neurotransmitters/hormones, noradrenaline and adrenaline, are unique to members of the deuterostome lineage and they have no physiological relevance in protostomes (including insects). In insects, their roles are fulfilled by their invertebrate counterparts, octopamine and tyramine (Roeder 2005). The biogenic amine octopamine in insects, and other invertebrates, carries out many of the physiological roles such as aggression (Zhou et al. 2008), locomotion (Koon et al. 2011; Wu et al. 2012), olfactory learning and memory (Farooqui et al. 2003; Schwaerzel et al. 2003; Unoki et al. 2005; Mizunami et al. 2009), ovulation (Monastirioti et al. 1996; Monastirioti 2003), and innate immunity (Adamo 2010; Huang et al. 2012).

In insects, the actions of octopamine and tyramine have been shown to be mediated via the activation of G-protein-coupled receptors. So far, many octopamine and tyramine receptors have been cloned from several insect species such as Drosophila melanogaster (Han et al. 1998; Cazzamali et al. 2005; Maqueira et al. 2005), Apis mellifera (Blenau et al. 2000; Grohmann et al. 2003), Bombyx mori (Ohta et al. 2003; Huang et al. 2009; Chen et al. 2010), and Chilo suppressalis (Huang et al. 2012; Wu et al. 2012, 2013). Based on the structural and signaling similarities between cloned D. melanogaster octopaminergic receptors and vertebrate adrenergic receptors, Evans and Maqueira proposed a new classification (Evans and Maqueira 2005). This classification has grouped insect octopamine receptors into three classes, namely, α1-adrenergic-like receptors (OA1), β-adrenergic-like receptors (OA2), and octopamine/tyramine or tyramine 1 receptors (TyR1) (Evans and Maqueira 2005; Verlinden et al. 2010). Later on, Cazzamali and co-workers cloned a gene (CG7431) from D. melanogaster that is specifically activated by tyramine, implying that it may belong to a new family of tyramine receptors (Cazzamali et al. 2005). An orthologous receptor was also characterized in Bombyx mori (Huang et al. 2009). Based on these findings, Farooqui made a revision in the new receptor classification by adding another subclass in the tyraminergic receptors, tyramine receptor type 2 (Farooqui 2012). A recent work showed a gene (CG16766) from D. melanogaster, which was activated by tyramine, has a different signal transduction and pharmacological profile to that of CG7431 (Bayliss et al. 2013). Besides inducing [Ca2+]i response, incubation CG16766-expressing Chinese hamster ovary-K1 cells with tyramine can also reduce forskolin-stimulated [cAMP]i. Hence, this gene have been designated the Tyramine 3 receptors (Bayliss et al. 2013).

In this study, we have isolated a new family of octopamine receptor gene (an orthologous gene of CG18208 in Drosophila) from Chilo suppressalis, viz., CsOA3. Two isoforms of the CsOA3 receptor are generated by differential splicing of the same gene and referred to as short (CsOA3S) and long (CsOA3L) octopamine receptors. Phylogenetic analysis clearly indicated that CsOA3 not belong to any known members of the invertebrate octopamine/tyramine receptor family. Using HEK293 cells stably transfected with CsOA3S and CsOA3L, respectively, we compared the cellular response to biogenic amines and selected synthetic agonists and antagonists. The results indicated that these two isoforms showed not identical second messenger cascades, pharmacological properties and tissue expression patterns. To our knowledge, this article is the first report on this novel family of insect octopamine receptors.

Materials and methods

Insects, tissue collection, and reagents

The larvae of the rice stem borer, C. suppressalis were reared on an artificial diet (Han et al. 2012) at 28°C under a photoperiod of 16:8 h (light:dark) for several generations prior to experiments. We collected various tissues of the rice stem borer as previously described (Wu et al. 2013). Briefly, hemocytes, fat body, midgut, Malpighian tubules, nerve cord, and epidermis of fifth-instar larvae were dissected under saline solution and immediately deep frozen in liquid nitrogen, and then stored at −80°C until treatment. For hemocytes collection, fifth-instar naïve larvae were surface sterilized with 70% ethanol and total hemolymph was collected by cutting its proleg. The entire nerve cord, including the brain, suboesophageal ganglion, thoracic ganglion, and abdominal ganglion, was dissected for RNA extraction. All of the reagents were purchased from Sigma-Aldrich (St Louis, MO, USA).

Gene cloning and phylogenetic analysis

Genomic DNA and total RNA was prepared from the C. suppressalis using DNeasy 96 Tissue Kit (Qiagen, Chatsworth, CA, USA) and TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol, respectively. Genomic DNA was used as template for amplifying the genomic DNA sequence of CsOA3. RNA was used as template for cDNA synthesis using the TransScript First-Strand cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China). We performed transcriptome sequencing of C. suppressalis and one putative octopamine receptor fragment (CsOA3) was annotated using BlastX [National Center for Biotechnology Information (NCBI), Bethesda, MD, USA]. The 5′- and 3′- ends of CsOA3 were obtained using 5′ Full RACE Kit and 3′ Full RACE Core Set Ver. 2.0 (both from TaKaRa, Dalian, China), respectively. The primers used in this study were showed in the Table S1. The cloned cDNA sequences were submitted to GenBank with the following accession numbers: CsOA3S, KF460458 and CsOA3L, KF460457.

To identify potential orthologs of the cloned C. suppressalis octopamine receptors, we constructed the phylogenetic trees of this receptor with other biogenic amine receptors. The receptor sequences were aligned using ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/). The tree was drawn using MEGA 5.0 with the maximum likelihood method (Tamura et al. 2011) and the branch support values are expressed as percentages. The tree was rooted using the sequence of the Drosophila FMRFamide receptor (CG2114). The accession number of sequences used in the study was shown in Table S2.

Cell culture, transfection, and generation of stable cell lines

The expression-ready construct of the CsOA3S and CsOA3L cDNA containing the Kozak consensus sequence (Kozak 1984) immediately 5′ to the initiating ATG codon was generated by PCR using the primers in Table S1. The PCR product was digested with Kpn1 and EcoR1 and subcloned into the pcDNA3 vector (Invitrogen) yielding pcDNA3-CsOA3S and pcDNA3-CsOA3L vector. Both of vectors were introduced into the HEK293 cells using Lipofectamine 2000 (Invitrogen). After 2 weeks of 800 μg/mL G418 (Invitrogen) selection, 16–24 G418-resistant colonies were trypsinized in cloning cylinders and transferred to 12-well plastic plates for expansion. These individual cell lines were analyzed for receptor expression by western blot and by immunocytochemistry. Stable cell lines were maintained at 37°C in 5% CO2 in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum and antibiotics (250 μg/mL G418, 100 U/mL penicillin, 100 μg/mL streptomycin). The clonal cell lines that most efficiently expressed CsOA3S and CsOA3L were chosen for this study.

cAMP determination

Ligand stimulation of cAMP production was monitored using the cAMP Parameter Assay Kit (R & D Systems, Minneapolis, MN, USA) as previously described (Wu et al. 2012, 2013). Briefly, the cells were plated into 24-well tissue culture plates (Nunc, Roskilde, Denmark) at a density of 5 × 105 cells well-1 and cultured at 37°C and 5% CO2 in a humidified incubator. The cells were washed in 1 × Dulbecco's phosphate buffered saline (D-PBS) (Gibco BRL, Carlsbad, CA, USA) and equilibrated for 20 min at 37°C in 100 μM of the phosphodiesterase inhibitor 3-isobutyl-1-methyl xanthine. After the pre-incubation, a 200 μl aliquot of D-PBS containing various concentrations of agonists was added, and then the culture was incubated for 20 min at 37°C. The reactions were stopped by removal of agonist solutions and the immediate lysis of the cells was stopped by addition of ice-cold cell lysis buffer. For antagonist studies, the stimulations were carried out as above except that the respective 10 μM antagonists were mixed with 1 μM octopamine. Values for each concentration tested were measured in duplicate three times.

Basal levels of HEK293 cells cAMP were 8.5 ± 0.1 pmols/well or 10.1 ± 0.2 pmols/well and these were raised to 23.9 ± 0.9 pmols/well or 46.2 ± 1.4 pmols/well after exposure to 10 μM forskolin in pcDNA3-CsOA3S or pcDNA3-CsOA3L HEK293 cells.

Calcium mobilization

Ligand stimulation of calcium elevation was measured as previously described (Huang et al. 2012). Briefly, CsOA3S- or CsOA3L-expressing HEK293 cells were seeded on the coverslip with Dulbecco's modified Eagle's medium and incubated for overnight at 37°C in 5% CO2. After incubation, the cells were subsequently washed twice with D-PBS and were loaded with Fura 2-AM (Dojindo Laboratories, Kumamoto, Japan) using 0.2% Cremophor EL (Sigma–Aldrich) for 30 min. The coverslip were transferred to a microscopic chamber that was constantly perfused with a bathing solution (152 mM NaCl, 5.4 mM KCl, 5.5 mM glucose, 1.8 mM CaCl2, 0.8 mM MgCl2, and 10 mM HEPES, pH 7.4) at approximately 2 mL/min. The fluorescence at 510 nm by excitation at 340 or 380 nm with a xenon lamp was measured with individual cells using an Easy Ratio Pro calcium imaging system (PTI, Birmingham, NJ, USA). A single coverslip of cells was used per concentration of agonist. Recordings were taken from up to 20 or at random when greater than 20 cells per field of view. The results from each coverslip gave an = 1 and each agonist concentration was repeated three times or more.

qPCR

For CsOA3 tissue expression, total RNA (1 μg) was digested with RQ1 RNase-Free DNase (Promega, Madison, WI, USA), and cDNA was synthesized with Rever Tra Ace qPCR RT kit (Toyobo, Osaka, Japan) according to the manufacturer's instructions. qPCR was performed on cDNA preparations using the SsoFast EvaGreen Supermix with Low Rox (Bio-Rad, Hercules, CA, USA) and Applied Biosystems 7500 real-time PCR system (Applied Biosystems by Life Technologies, Carlsbad, CA, USA). Using the 2-ΔΔCt method, the data are presented as the fold change in CsOA3 gene expression normalized to the elongation factor 1 (EF-1) gene (endogenous control). The primers are provided in Table S1, and each reaction was performed in triplicate.

Statistical analysis

Numbers in histograms represent mean ± SEM. Statistical comparisons were done using the anova with Tukey–Kramer post hoc test (*< 0.05, **< 0.001, ***< 0.0001). All curve fitting and statistical calculations were performed with Origin 8.0 (Origin Lab, Northampton, MA, USA). EC50 is the agonist concentration that evoked the half-maximal response.

Results

Molecular features of cloned CsOA3 receptors

The full length of genomic DNA and cDNA of CsOA3 was amplified from C. suppressalis by using transcriptome analysis with a polymerase chain reaction (PCR)-based strategy. The CsOA3 gene produces two isoforms, CsOA3S and CsOA3L, which are generated by alternative splicing (Fig. 1a) (Figure S1). The ORF of CsOA3L (1653 bp) encodes a protein of 550 amino acids with a calculated molecular weight of 61.5 kDa. CsOA3L differs from CsOA3S by the presence of an additional 30 amino acids within the third intracellular loop (Fig. 1b, c). This region is implicated in the receptor interaction with G-proteins (Usiello et al. 2000). Interestingly, according to the genomic and transcriptional data, we found that the orthologous genes of CsOA3 in D. melanogaster and T. castaneum also have both long and short transcripts by alternative splicing between TM5 and TM6, same as C. suppressalis (Fig. 1c). Therefore, it seems that this kind of alternative splicing in the third intracellular loop of CsOA3-like genes could be conserved among different insect species. Amino acid sequence comparisons between CsOA3 and other insect biogenic amine receptors show high overall amino acid similarity (identical and conservatively substituted amino acid) to predicted octopamine receptors of D. melanogaster (CG18208; 79%; NCBI accession number: NP_001262714), and Tribolium castaneum (similar to CG18208 CG18208-PA; 70%; NCBI accession number: XP_969656). We also sequence motifs, which are essential for the ligand binding and signal transduction of the receptor, are well conserved in CsOA3 (Fig. 1c). Among these are the tripeptide D-R-Y (Asp174-Arg175-Tyr176) motif at the end of TM3, which is well conserved among family A G-protein-coupled receptors (GPCRs) and is believed to be involved in receptor activation (Rovati et al. 2007). Consensus motifs for phosphorylation by protein kinase A (PKA) and C (PKC) are important for receptor desensitization and internalization (Ferguson 2001). Four or five consensus sites for phosphorylation by PKC are found within the third intracellular of CsOA3S or CsOA3L, respectively. N-linked glycosylation sites were found in the N-terminus of CsOA3. Two conserved cysteines are located in extracellular loops 1 and 2, which have been suggested to stabilize receptor structure by forming disulfide bonds (Rader et al. 2004). CsOA3 has a large third intracellular loop and a short C-terminus, which is also a common feature of the TyR1 family (Wu et al. 2013). The C-terminus of CsOA3 consists of only 14 amino acids. It lacks cysteine residues, which are possible targets for palmitoylation and therefore cannot become palmitoylated like other GPCRs (Qanbar and Bouvier 2003).

Figure 1.

Molecular cloning and multiple sequence alignment of the two isoforms of CsOA3 receptor. (a) The two molecular isoforms of CsOA3 receptor mRNAs detected by the polymerase chain reaction (PCR). (b) Schematic representation of the CsOA3 receptor genomic and the two molecular mRNA isoforms detected by the PCR. The top lines represent the original genomic sequences. It flanks the shaded region X in CsOA3S and the 30 amino acids were shown. In the predicted topologies of the receptors the transmembrane regions are indicated as 1, 2, 3, 4, 5, 6, and 7. (c) Amino acid sequence alignment of CsOA3S/L and orthologous receptors from Drosophila melanogaster (DmOA3S/L; CG18208, no. NM_142497.3) and Tribolium castaneum (TcOA3S/L; no. XP_969656.2). The amino-acid position is indicated on the right. Identical residues between CsOA3S/L and any of the aligned sequences are shown as white letters against black, whereas conservatively substituted residues are shaded. Dashes indicate gaps that were introduced to maximize homologies. The predicted seven transmembrane regions are indicated by TM 1–7. Potential N-glycosylation sites (●) and potential phosphorylation sites (*) for protein kinase C are labeled. Conserved cysteine residues in the 1st and 2nd extracellular loop are labeled (○). The aspartic acid residue (D157) and the serine residues (S240 and S244) that are predicted to be involved in agonist binding are labeled with filled triangles and quadrilateral, respectively. The second phenylalanine (◊) after the FxxxWxP motif in TM6 is a unique feature of aminergic receptors. The arrow (G384) indicates the position of alternative splicing of the CsOA3 gene.

To identify the evolutionary relationships of the cloned C. suppressalis octopamine receptors with others biogenic amine receptors, and in that way to provide evidence for their possible functional roles, a phylogenetic analysis was performed together with human catecholamine receptors and invertebrate biogenic amine receptors. The analysis clearly shows that CsOA3S/L receptor clustered with CG18208 (DmOA3), AmOA3, and TcOA3 in a distinct clade, which indicated that it belongs to a new family of octopamine/tyramine receptors. A closely related clade contained the human α2-adrenergic receptors (Fig. 2).

Figure 2.

Phylogenetic analysis of CsOA3S/L and other selected invertebrate biogenic amine receptors. Maximum likelihood trees were constructed using MEGA 5 software with 1000-fold bootstrap re-sampling. The numbers at the nodes of the branches represent the level of bootstrap support for each branch. Drosophila melanogaster FMRF (DmFR) amide receptor was used as an outgroup. For accession numbers to the used sequences, see Table S2.

Functional expression studies on the coupling of CsOA3S and CsOA3L to adenylyl cyclase activity

Biogenic amine specificity

We compared the abilities of related biogenic amines to alter intracellular [cAMP]i in HEK293 cell lines stably expressing either the CsOA3S or the CsOA3L putative octopamine receptor. Both of them were screened for their abilities to increase [cAMP]i in response to a wide range of naturally occurring biogenic amines (octopamine, tyramine, dopamine, serotonin) tested at a concentration of 1 μM. It can be seen that four biogenic amines had no significant effects on cAMP production in both CsOA3S- and CsOA3L-expressing cells (Fig. 3a, b). Furthermore, we examined the effects of four biogenic amines on the forskolin-stimulated cAMP production. Both octopamine and tyramine significantly attenuated the forskolin-simulated production of cAMP in CsOA3S-expressing, but not in CsOA3L-expressing HEK293 cells (Fig. 3a, b). Statistical analysis also showed that there is a significant difference between the responses of CsOA3S to octopamine and tyramine when applied them at concentration of 1 μM (Fig. 3a). All of four biogenic amines had no effects on cAMP production and forskolin-stimulated cAMP production in non-transfected HEK293 cells at a concentration of 1 μM. The dose–response relationships of octopamine and tyramine on the [cAMP]i were examined in CsOA3S-expressing cells. The effects of both compounds were concentration-dependent and saturable, resulting in a sigmoidal dose–response curve (Fig. 3c). Half-maximal reduction of cAMP production (EC50) was achieved at a octopamine concentration of 3.9 × 10−8 M and tyramine concentration of 9.4 × 10−8 M (Table 1) with threshold concentrations for both amines occurring between 1 and 10 10−9 M for an effect on forskolin-stimulated [cAMP]i. Moreover, tyramine showed lower efficacy (reduced to 43.5% of the maximum effects) than that of octopamine (reduced to 56.2% of the maximum effects). Thus, octopamine is more potent and efficacious than tyramine to active CsOA3S.

Figure 3.

Effects of biogenic amines and agonists on intracellular cAMP levels in CsOA3S-expressing (a, c) and CsOA3L-expressing (b) HEK293 cells. For (a and b), results are expressed as the fold increase of cAMP relative to untreated cells. For (c), the amount of cAMP is given as the percentage of the value obtained with 10 μM forskolin (= 100%). Data are expressed as the mean ± SEM (> 3). Asterisks indicate values significantly different from the control value using one-way anova with the Tukey-Kramer multiple comparisons test (***p < 0.0001).

Table 1. cAMP formation and calcium mobilization in HEK293 cells expressing CsOA3S or CsOA3L induced by various agonists in a concentration-dependent manner
AgonistsCsOA3SCsOA3L
cAMPCa2+cAMPCa2+
EC50 (M)EC50 (M)Emax (%)EC50 (M)EC50 (M)Emax (%)
  1. The EC50 values and the maximum effects (relative Ca2+ flux to naphazoline) shown are based on > 3 independent experiments. Data are presented as means ± SEM.

  2. ND, not determined.

Octopamine3.9 ±  (0.29) × 10−81.0 ±  (0.19) × 10−990.5 ± 2.2ND5.7 ±  (0.06) × 10−1095.8 ± 2.2
Tyramine9.4 ±  (1.64) × 10−83.2 ±  (0.12) × 10−977.8 ± 2.4ND1.9 ±  (0.55) × 10−969.8 ± 2.2
DopamineND3.2 ±  (0.11) × 10−975.8 ± 2.7ND3.1 ±  (0.16) × 10−969.5 ± 2.3
Naphazoline2.3 ±  (0.71) × 10−112.5 ±  (0.07) × 10−1096.3 ± 5.2ND8.7 ±  (0.08) × 10−1198.6 ± 9.9
Clonidine5.0 ±  (0.54) × 10−123.7 ±  (0.06) × 10−1096.8 ± 3.4ND4.9 ±  (0.08) × 10−1194.6 ± 5.4

Effects of synthetic agonists and antagonists

In order to determine the pharmacological profile of CsOA3S, we examined the effects of various potential octopamine receptor agonists and antagonists. It can be seen that naphazoline and clonidine both showed concentration-dependent decreases in forskolin-stimulated cAMP levels in CsOA3S-expressing cells. The rank order of potency (measured as EC50) was: clonidine (5.0 × 10−12 M) > naphazoline (2.3 × 10−11 M), and clonidine was found to be ten thousand times more potent than octopamine. Control experiments indicated that none of the synthetic agonist tested had any effects on forskolin-stimulated cAMP levels in non-transfected wild-type HEK293 cells (data not shown). Thus, α-adrenergic agonist seems to be very effective on CsOA3S. Furthermore, the ability of putative antagonists to impair the octopamine-induced attenuation of cAMP synthesis was examined (Fig. 4). The effects of octopamine could be significantly blocked by co-incubation with the typical α-adrenergic antagonists phentolamine (= 0.0035) or the epinastine (= 0.000675). In contrast, the α2-adrenergic antagonists yohimbine and mianserin, and the dopaminergic antagonists, chlorpromazine, SCH 23390, and flupenthixol, had no significant blocking effect on the receptor.

Figure 4.

Effects of putative antagonists (10 μM) on OA attenuation of forskolin-stimulated intracellular cAMP levels in CsOA3S-expressing HEK293 cells. Asterisks indicate statistically significant differences for the treatments versus 10 μM of octopamine alone. Data represent means ± SEM of four to six experiments. Asterisks indicate values significantly different from the control value using one-way anova with the Tukey–Kramer multiple comparisons test (***p < 0.0001). Abbreviations: OA, octopamine and no antagonist; OA/YH, 10 μM of octopamine + 10 μM of yohimbine; OA/CH, 10 μM of octopamine + 10 μM of chlorpromazine; OA/PA, 10 μM of octopamine + 10 μM of phentolamine; OA/MS, 10 μM of octopamine + 10 μM of mianserin; OA/EP, 10 μM of octopamine + 10 μM of epinastine; OA/SCH, 10 μM of octopamine + 10 μM of SCH 23390; OA/FLU, 10 μM of octopamine + 10 μM of flupenthixol.

Functional expression studies on the coupling of CsOA3S and CsOA3L to intracellular calcium mobilization

The above four biogenic amines and two agonists were further tested for their ability to generate intracellular Ca2+ signaling. Serotonin did not produce Ca2+ responses in both CsOA3S and CsOA3L receptors (data not shown). While addition of octopamine, tyramine, and dopamine to CsOA3S- or CsOA3L-expressing HEK293 cells resulted in concentration-dependent increases in intracellular Ca2+ levels (Fig. 5). We constructed full dose–response curves for the biogenic amines, naphazoline and clonidine (Fig. 5a, b). It can be seen that the rank order of potency was different for each of the receptors [EC50s: CsOA3S naphazoline (2.5 × 10−10 M) > clonidine (3.7 × 10−10 M) > octopamine (1.0 × 10−9 M) > dopamine (3.2 × 10−9 M) > tyramine (3.2 × 10−9 M); CsOA3L clonidine (4.9 × 10−11 M) > naphazoline (8.7 × 10−11 M) > octopamine (5.7 × 10−10 M) > tyramine (1.9 × 10−9 M) > dopamine (3.1 × 10−9 M)] (Table 1). However, the maximum responses compared with naphazoline were 90.3% or 95.8% for octopamine, 79.1% or 69.8% for tyramine and 79.8% or 69.5% for dopamine in CsOA3S- or CsOA3L-expressing HEK293 cells (Fig. 5a, b). Hence, tyramine and dopamine appeared to be partial agonists of CsOA3S and CsOA3L, while octopamine appeared to be a full agonist.

Figure 5.

Dose-dependent increases in [Ca2+]i in HEK293 cells transfected with the CsOA3S (a) and CsOA3L (b) receptor after exposure to different concentrations of biogenic amines and synthetic agonists. Means ± SEM from three to five experiments are shown after normalization of the Ca2+ responses in relation to the maximum response obtained with naphazoline.

We also performed experiments with nominally no Ca2+ in the extracellular solution (Ca2+ was substituted for 20 mM EGTA). Application of 1 μM octopamine again resulted in a single Ca2+ signal in both of CsOA3S- or CsOA3L-expressing HEK293 cells (Figure S2). It indicated that CsOA3 receptors couple to the phospholipase C/inositol triphosphate signaling pathway to cause Ca2+ release from intracellular stores. No Ca2+ response was observed in non-transfected cells when treated with 1 μM of four biogenic amines (Figure S3).

Differential expression of the C. suppressalis octopamine receptor

To compare the mRNA expression levels of cloned C. suppressalis receptors, CsOA3 and CsOA3L, quantitative real-time PCR was performed in the fifth-instar larval stage. It was found that this gene was expressed in all tested tissues, including hemocytes, fat body, midgut, Malpighian tubules, nerve cord, and epidermis (Fig. 6). CsOA3 has two isoforms, CsOA3S and CsOA3L, which are generated by alternative splicing and co-expressed in a ratio favouring the short isoform, CsOA3S (Fig. 6). It should be noted that the primers which amplified CsOA3 contain two isoforms. The expression level of the CsOA3 and CsOA3L gene was higher in the Malpighian tubules and nerve cord than in other tested tissues, suggesting that it might play an important role in the excretory system and central nervous system of insects.

Figure 6.

mRNA expression of the cloned C. suppressalis receptors. Expression pattern of CsOA3 and CsOA3L mRNA levels in tissues of fifth-instar larvae were quantified by qPCR. Tissues tested are hemocytes (HC), fat body (FB), midgut (MG), Malpighian tubules (MT), nerve cord (NC) and epidermis (EP). Data represent means ± s.e.m (> 3).

Discussion

Cloning and classification of CsOA3

In this study, we have cloned and functionally characterized an octopamine receptor from the rice stem borer, C. suppressalis. Orthologous receptors have not been isolated from other invertebrate species. Structural and pharmacological functional studies demonstrated that this gene belong to a novel family of octopamine receptors, which we named it CsOA3 receptor. The CsOA3 codes for two polypeptides, CsOA3S and CsOA3L, those are generated by alternative splicing. CsOA3L differs from CsOA3S by the presence of an additional 30 amino acids within the third intracellular loop. A phylogenetic analysis of the CsOA3S and CsOA3L receptor sequences clearly shows that both receptor sequences group together with CG18208 in D. melanogaster and two CG18208-like receptor genes in the genomic database from the beetle T. castaneum and the honeybee A. mellifera. Interestingly, we found that this mechanism of splicing was conserved in D. melanogaster and T. castaneum, as the model insects, their genome sequences are known. It was reported that there are four families of octopamine receptors and tyramine receptors, including α1-adrenergic-like octopamine receptors (OA1), β-adrenergic-like octopamine receptors (OA2), TyR1 and tyramine receptor type 2 (Evans and Maqueira 2005; Farooqui 2012). CsOA3 is different from known octopamine and tyramine receptors, and has structural similarities with vertebrate α2-adrenergic receptors and insect TyR1. Hence, our finding favor a revision in the new receptor classification by Farooqui (2012) and Evans and Maqueira (2005), by adding another subclass, α2-adrenergic-like octopamine receptors (OA3), in the octopaminergic class of receptors as shown in Fig. 7.

Figure 7.

Classification schemes of octopaminergic receptors. A new receptor classification based on cloning and functional studies of third class of octopaminergic receptors. Abbreviations: Ca2+, calcium; cAMP, cyclic adenosine monophosphate.

Downstream signaling of CsOA3 receptors

The type of intracellular signaling that ensues GPCR activation depends on the associated G-protein, most importantly the Gα subunit. Coupling with Gαs activates adenylyl cyclase that convert ATP to cAMP, whereas coupling with Gαi and Gαo suppresses adenylyl cyclases activity. Gαq-containing G-proteins interact with phospholipase C, leading to a signaling cascade that ends with an increase [Ca2+]i levels (Park and Adams 2005). In contrast to the well investigated mammalian G-proteins, knowledge about the G-protein families of invertebrates is still rather limited (Lind et al. 2010). It was reported that coupling with specific G-proteins is brought about by amino acids in close vicinity to the plasma membrane of the second and third intracellular loops and of the cytoplasmic tail of the receptor proteins. Biogenic amine receptors that couple to Gαi-proteins and suppress adenylyl cyclase activity often possess short C termini and long third intracellular loop (Probst et al. 1992; Rotte et al. 2009). This feature is conserved in CsOA3 and insect TyR1 family, which indicated that CsOA3 might have a similar signaling pathway as TyR1 family. Besides, insect α1-adrenergic-like octopamine receptors, which coupled to both Ca2+ and [cAMP]i increases, have a long third intracellular loop (Ohtani et al. 2006). However, insect β-adrenergic-like octopamine receptors and D1-like dopamine receptors have a short third intracellular loop and both families selectively coupled to the production of cAMP (Beggs et al. 2011; Wu et al. 2012).

CsOA3S and CsOA3L were stably expressed in HEK293 cells, which have been used successfully in previous studies to examine the pharmacological properties of cloned insect biogenic amine receptors (Balfanz et al. 2005; Maqueira et al. 2005; Wu et al. 2012). Activation of CsOA3S by octopamine led to decrease forskolin-stimulated [cAMP]i (Fig. 4). However, this effect has not been observed in CsOA3L-expressing cells activated by octopamine. In addition to [cAMP]i signaling, activation of both CsOA3S and CsOA3L by octopamine led to Ca2+ release from intracellular stores. Hence, the new classification was supported by our functional studies because both CsOA3S and CsOA3L were activated by octopamine with high efficacy and potency. They were also activated by tyramine but with lower potency and efficacy.

Insect TyR1 family has been reported to couple with two different signaling pathways, that is, DmTyR1 expressed in Chinese hamster ovary cells (Robb et al. 1994), Xenopus oocytes (Reale et al. 1997), or Drosophila Schneider 2 cells (Essam 2005); the locust LocTyR1 receptor expressed in murine erythroleukemia cells (Poels et al. 2001) have been shown to increase the intracellular Ca2+ concentration except to inhibit adenylyl cyclase activity. However, some reports indicated that there is no increase in intracellular Ca2+ concentration when activated by tyramine (1 μM) in AmTyR1 (Blenau et al. 2000) and PeaTyR1 (Rotte et al. 2009). Compared with insect TyR1 family, the activation of CsOA3S is also coupled with not only Gi-protein that inhibits adenylyl cyclase but also with Gq-protein that elevates intracellular Ca2+ levels. Moreover, octopamine and tyramine decrease forskolin-stimulated [cAMP]i at a very low concentrations (octopamine, EC50 = 3.9 × 10−8 M; tyramine, EC50 = 9.4 × 10−8 M). Whereas in insect TyR1 family which was activated by tyramine, the EC50 values were 10-fold higher than that in CsOA3S [CsTyR1: EC50 = 3.7 × 10−7 M (Wu et al. 2013); PeaTyR1: EC50 = 3.5 × 10−9 M (Rotte et al. 2009); AmTyR1: EC50 = 1.3 × 10−9 M (Blenau et al. 2000)]. With respect to Ca2+ response, octopamine and tyramine also showed very high potency in CsOA3 (Table 1).

Pharmacological properties of CsOA3 receptors

CsOA3 showed a distinct pharmacology to reported octopamine and tyramine receptors in terms of both its agonist-mediated [cAMP]i and Ca2+ responses. Regards synthetic agonists, CsOA3 showed some similarities with the β-adrenergic-like octopamine receptors in Drosophila (Maqueira et al. 2005), Bombyx (Chen et al. 2010) and Chilo (Wu et al. 2012) in that it was activated by naphazoline and clonidine. It also showed similarities with Bombyx OA1 receptor (Huang et al. 2010) when considering Ca2+ responses.

A range of seven potential antagonists were examined for their antagonism toward OA-induced [cAMP]i decreases in CsOA3S-HEK cells. A phylogenetics analysis of CsOA3 shows that it groups together with human α2-adrenergic receptors, suggesting α2-adrenergic-blockers may also act on this receptor family. However, yohimbine, an α2-adrenergic receptor antagonist which has been shown to have antagonistic effects on insect TyR1 (Saudou et al. 1990; Rotte et al. 2009; Wu et al. 2013), was not able to block the actions of octopamine on CsOA3S. While two antagonists, phentolamine and epinastine, were found to significantly reduce octopamine-stimulated adenylyl cyclase inhibition. We previous reported that phentolamine, which is generally known as an antagonist for octopamine receptors, also displayed antagonistic effects on CsOA2B2 (Wu et al. 2012). Epinastine was been found to be a highly selective antagonist for insect octopamine receptors (Roeder et al. 1998). It is reported that co-incubation of octopamine together with epinastine abolished the octopamine-induced cAMP production in locust nervous tissue homogenates. However, it was ineffective on CsOA2B2 (Wu et al. 2012) and had a weak antagonist effect on BmOAR1 (Huang et al. 2010). Our results indicated that the OA3 receptor family might be the target of this antagonist. In addition, chlorpromazine and mianserin, which have antagonistic effects on β-adrenergic-like octopamine receptors (Wu et al. 2012), did not show any inhibition effect. Thus, CsOA3 showed an agonist profile similar to that of β-adrenergic-like octopamine receptors but the antagonist profile did not match.

Functional implications of CsOA3 receptors

Compared with the insect OA3 receptor family, the mice dopamine D2 receptor gene also encodes two molecular distinct isoforms, D2S and D2L, and these receptors have distinct functions in vivo. D2L acts mainly at post-synaptic sites and D2S serves presynaptic autoreceptor functions (Usiello et al. 2000). It would thus appear that in insects octopamine could bring about its physiological actions by potentially interacting not only with two or three groups of octopamine receptors but also with two isoforms of one receptor. To examine the exact physiological roles of OA3S and OA3L in different locations in the insect nervous system, and in other tissues, it will be necessary to investigate their tissue-specific and stage-specific expression patterns. However, it could also be a multifunctional receptor that is activated by octopamine at some locations and by tyramine at other locations, depending upon the identity of the amine released presynaptically.

Acknowledgements

This study was supported by National Program on Key Basic Research Projects (973 Program, 2013CB127600), National High-tech R&D Program of China (2011AA10A204) and National Special Agricultural Research Projects for Public Welfare, China (201303017). The authors thank Pi-Hua Zhou, Gu-Qian Wang and Shuang-Yang Wu for assistance in collecting the rice stem borer samples. The authors declare no conflicts of interest.

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